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.     

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.     

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.     

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.     

Tam3 in Antirrhinum majus is exceptional transposon in resistant to alteration by abortive gap repair: identification of nested transposons

Most transposon families consist of heterogeneous copies with varying sizes. In contrast, the Tam3 copies in Antirrhinum majus are known to have exceptionally conserved structures of uniform size. Gap repair has been reported to be involved in the structural alteration of copies from several transposon families. In this study, we have asked whether or not gap repair has affected Tam3 copies. Five Tam3 copies carrying aberrant sequences were selected from 40 independent Tam3 clones and their sequences were analyzed. Two of the five copies contain insertions in the Tam3 sequence. These two insertions, designated Tam356 and Tam661, are typical transposon-like sequences, which have terminal inverted repeats and cause target site duplication. These nested transposons were obviously associated with transpositional events, and did not originate from the gap-repair process. The remaining three copies had lost large parts of the Tam3 sequence. We could not find any relationship between the deletions of Tam3 sequence in the three copies and gap repair. PCR analysis of a Tam3 excision site in the nivea ^{recurrence:Tam3} mutant also showed that most of the repair events after the Tam3 excision involved end-joining. In addition to the results obtained here, among the other clones isolated, we could not find any of the internally deleted copies that comprise a major part of other transposon families. All of these data suggest that some feature of the Tam3 structure suppresses the structural alterations that are otherwise generated during the gap repair process.     

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.     

The Spm (En) transposable element controls the excision of a 2-kb DNA insert at the wx allele of Zea mays

The waxy (Wx) locus of Zea mays was cloned from strains carrying the wild-type and wx^m-8 mutant alleles. The receptor component of the Suppressor-Mutator (Spm) controlling element system in the wx^m-8 allele was shown to be a 2 kb long insertion within the transcribed region of the Wx gene. The insertion, termed Spm-I8, is excised during somatic reversion events induced by the autonomous controlling element Enhancer (En), which is an equivalent to Spm. Integration of Spm-I8 into the Wx gene generates a 3-bp target site duplication. Spm-I8 has a 13 bp long inverted repeat at its termini. The ends of the element can be further folded to build a large double-stranded structure consisting of five perfectly matching double-stranded regions of 9–13 bp in length, interrupted by single-stranded loops. A comparison of the wild-type and wx^m-8 alleles revealed two additional insertions 6 (insert-1) and 0.25 (insert-2) kb in length. No En-induced excision of insert-1 and insert-2 could be detected so far. There is remarkable structure and sequence homology between Spm-I8 and the transposable elements Tam1 and Tam2 of Antirrhinum majus at their termini, reflecting a possible evolutionary and/or functional relationship between transposons in different plant species.     

The structure of hobo transposable elements and their insertion sites

The hobo transposable elements of Drosophila form a family of 3.0-kb elements and their deletion derivatives. Their distribution is consistent with the model that 3.0-kb elements are functionally complete but that smaller hobos are defective and require complete elements in trans for transposition. The sequence of one 3.0-kb element is presented; it has several interesting features, including a 1.9-kb open reading frame downstream from potential TATA and CAT sequences. Comparison of 11 independent insertion sites shows that in every case the hobo element has integrated at and duplicated either the sequence NNNNNNAC or CTTTNNNN. There is evidence that an eight nucleotide sequence internal to hobo that matches both of these sequences has been used as an insertion site for a second hobo element, as the first step in the creation of an internal deletion derivative. Structural similarities between hobo and the eukaryotic transposable elements P, Ac, 1723, and Tam3, found in widely divergent host organisms, suggest that they all transpose by a common mechanism.     

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     

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.     

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.     

Strains of the Morphospecies Ploeotia costata (Euglenozoa) Isolated from the Western North Pacific (Taiwan) Reveal Substantial Genetic Differences

Two phagotrophic euglenid strains (Strains Pac and Tam) were isolated from coastal locations in Taiwan. Ultrastructural characteristics of the strains included five pellicle strips joined at the posterior end. The strips were formed by major grooves with bifurcated edges. At the cell anterior, the feeding structure formed a lip. Underneath the lip was a comb composed of layers of microtubules. Farther back, two supporting rods tapered toward the posterior end, and a number of vanes with attached microtubules were present between the rods. The morphological characteristics agree with Ploeotia costata Strain CCAP 1265/1. However, the 18S rDNA sequences of Strains Pac/Tam lacked a group I intron and possessed three extra insertions of 116, 67, and 53 bp. Phylogenetic analysis indicated low sequence similarity between Strains Pac/Tam and CCAP 1265/1 (92%). The morphospecies P. costata apparently includes a substantial level of DNA sequence divergence, and likely represents multiple molecular species units.     

Comparison of genetic behaviour of the transposable element Tam3 at two unlinked pigment loci in Antirrhinum majus

Summary In Antirrhinum majus the transposable element Tam3 has been described at two unlinked loci pallida and nivea, both of which are required for the production of anthocyanin pigment in flowers. In each case the element is inserted in the promoter region and gives a variegated phenotype. We show that the rate of Tam3 excision at both loci is greatly affected by temperature, being approximately 1000-fold higher at 15°C compared with 25°C. Tam3 is also controlled by an unlinked gene Stabiliser, which considerably reduces excision rate. We show that the high degree of sensitivity to temperature and Stabiliser is an intrinsic property of Tam3 which is not shared by an unrelated element, Tam1. The Tam3 insertion at nivea gives rise to a series of alleles which confer reduced pigmentation, novel spatial patterns and changed instability. These are probably a result of imprecise excision and rearrangements of the Tam3 element.     

De novo activation of the transposable element Tam2 of Antirrhinum majus

Summary The nivea locus of Antirrhinum majus encodes the enzyme chalcone synthase required for the synthesis of red anthocyanin pigment. The stable allele niv-44 contains an insertion in the nivea gene (Tam2) which has all the structural features of a transposable element. We have shown that this insertion can excise from the nivea locus when niv-44 is combined with another allele (niv-99) in a heterozygote. Activation of Tam2 excision is caused by a factor tightly linked to the niv-99 allele and may be due to complementation between Tam2 and a related element, Tam1. Factors which repress the excision of Tam2 and Tam1 are also described. Repression is not inherited in a simple mendelian way. Many stable mutations may be due to the insertion of transposable elements. Our data suggest that their stability may be due to the absence in the genome of activating factors and to the presence of repressors.     

Binding ofNicotiana nuclear proteins to the subterminal regions of theAc transposable element

Specific binding ofNicotiana nuclear protein(s) to subterminal regions of theAc transposable element was detected using gel mobility shift assays. A sequence motif (GGTAAA) repeated in both terminal regions ofAc, was identified as the protein binding site. Mutation of two nucleotides in this motif was sufficient to abolish binding. Based on a series of competition assays, it is deduced that there is cooperative binding between two repeats, each similar to the GGTAAA motif. The binding protein is probably similar to a previously characterized maize protein which binds to a GGTAAA-containing motif located in the ends ofMutator. Moreover, we show that DNA fromDs1 competes for protein binding toAc termini, and we show, by sequence analysis, that GGTAAA binding sites are present in the terminal region ofTgm1, Tpn1, En/Spm, Tam3 andDs1-like elements. This suggests that the binding protein(s) might be involved in the transposition process.     

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.     

A new hobo,Ac, Tam3 transposable element,hopper, from Bactrocera dorsalis is distantly related to hobo and Ac

A new transposable element from the hobo, Ac, Tam3 transposon family was isolated as a genomic clone from the Oriental fruit fly, Bactrocera dorsalis. It is approximately 3.1 kb in length with 19-bp inverted terminal repeat sequences having a single mismatch. Though sharing several amino acid sequence identities with other hAT elements, it is distantly related to both hobo and Ac. Among hAT elements thus far described in insects, it is apparently the most distantly related to hobo.     

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.