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.     

Transposition of IS2 into the hemB gene of Escherichia coli K-12.

Genetic studies of the hemB gene in Escherichia coli have resulted in the recovery of both stable and unstable mutant strains. The stable strains have been shown to result from large deletions. This study demonstrates that unstable strains result from the insertion of transposable element IS2 primarily into the 5' region of the structural gene; the instability results from precise excision of the element, producing strains with both high and low frequencies of reversion. This first report of IS2 insertion into hemB suggests that this gene may be a preferred target for insertion of this transposable 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.     

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.     

Somatic Variegation and Germinal Mutability Reflect the Position of Transposable Element Dissociation within the Maize R Gene

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.     

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.     

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.     

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.     

Detection of de novo insertion of the medaka fish transposable element Tol2

Tol2 is a terminal-inverted-repeat transposable element of the medaka fish Oryzias latipes. It is a member of the hAT (hobo/Activator/Tam3) transposable element family that is distributed in a wide range of organisms. We here document direct evidence for de novo insertion of this element. A Tol2 clone marked with the bacterial tetracycline-resistance gene was microinjected into fertilized eggs together with a target plasmid, and the plasmid was recovered from embryos. The screening of plasmid molecules after transformation into Escherichia coli demonstrated transposition of tet into the plasmid and, by inference, precise insertion of Tol2 in medaka fish cells. De novo excision of Tol2 has previously been demonstrated. The present study provides direct evidence that the Tol2 element has the entire activity necessary for cut-and-paste transposition. Some elements of the mariner/Tc1 family, another widespread group, have already been applied to development of gene tagging systems in vertebrates. The Tol2 element of the hAT family, having different features from mariner/Tc1 family elements, also has potential as an alternative gene tagging tool in vertebrates.     

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.     

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.     

Molecular Analysis of a Transposon-Induced Deletion of the Nivea Locus in Antirrhinum Majus

The transposable element Tam3 of Antirrhinum majus is capable of causing large-scale chromosomal restructuring. It induced a large deletion at the nivea locus, to produce the allele niv-:529. The deletion removed the entire nivea coding region while the element remains intact with the potential to induce further rearrangements. Genetic experiments showed that the endpoint of the deletion (called x) is closely linked to nivea. The DNA sequences of niv-:529, a genomic excision of Tam3 from niv-:529, and the original genomic position of x have been determined. These data suggest that the deletion could have resulted from an abortive transposition or through breakage and religation.     

Molecular Analysis of the Maize Wx-B3 Allele Indicates That Precise Excision of the Transposable Ac Element Is Rare

The somatic and germinal behavior of the maize wx-B3 mutation indicates that this Ac allele rarely reverts. Endosperms containing wx-B3 display tiny and infrequent Wx revertant sectors while no significant reversion is detected when wx-B3 pollen is stained with I/KI. Previous studies of other transposable element alleles that revert infrequently have implicated low levels of element excision. Unlike these other alleles, the wx-B3 Ac element is indistinguishable from fully active Ac elements with respect to its structure, and its ability to transpose from the Wx gene or to trans-activate a Ds element. Characterization of somatic and germinal excision events lead us to conclude that excision of the wx-B3 Ac element almost always produces null alleles. Furthermore, the excellent correlation between the position of the wx-B3 mutation on the physical and genetic maps indicates that the Ac insertion is the only lesion of wx-B3. As a result, precise excision of this Ac should restore Wx function. The fact that revertant sectors and pollen grains are rare indicates that precise excision of Ac is also rare. The finding that the wx-B3 reversion frequency is comparable whether wx-B3 is hemizygous or over a wx allele with a wild-type insertion site illustrates a fundamental difference between the excision mechanisms of Ac and Drosophila P elements.     

Position effect of the excision frequency of the Antirrhinum transposon Tam3: implications for the degree of position-dependent methylation in the ends of the element

We identified eight independent Tam3 copies residing in the same Antirrhinum majus genome. All the copies showed excision at 15 °C, but not at 25 °C. Under conditions promoting excision, each copy appeared to transpose in the leaves and flower lobes with a nearly constant frequency, whereas individual transposition abilities varied widely: the most active copy had an excision frequency more than 100-fold greater than that of the least active one. Despite the different transposition abilities, the structures of the eight Tam3 copies were almost identical. These results made it clear that the transpositional ability of Tam3 is regulated by chromosomal position, but they do not imply position-dependent transposase activity. The position effect of the Tam3 transposition was found to be correlated to the methylation state of the copy's end regions: DNA methylation in the Tam3 end regions tended to suppress the excision activity, and the degree of methylation was dependent on the chromosomal position. Our results also provide evidence of de novo methylation provoked by transposition of the endogenous element. We propose a mechanism of transpositional regulation of plant transposons that responds to the degree of methylation as determined by chromosomal position.