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.     

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.     

The homeotic Macho mutant of Antirrhinum majus reverts to wild-type or mutates to the homeotic plena phenotype

Plants of Antirrhinum majus carrying the semidominant Macho alleles of the plena gene display carpelloid sepals and staminoid petals, but the two inner flower whorls of stamens and carpels are normal and produce fertile gametes. In the recessive plena mutant, in contrast, the two outer whorls are normal whereas the stamens are largely or entirely petaloid and the carpels sepaloid, thus producing weakly male-fertile or fully sterile lines. Two new plena and two new Macho alleles have been induced in transposon tagging experiments. Genetic and molecular analysis revealed that the two contrasting mutant phenotypes are caused by mutations in one and the same gene: Several wild-type plants appeared among 27 000 F1 plants of a cross between Macho female plants and wild-type males bearing the active transposons Taml and Tam3. One of these plants segregated plena mutants, three showed reversions to wild-type and another two segregated Macho plants, possibly representing somatic reversions. Additional evidence was provided by an allelism test of Macho × plena. Molecular analysis has independently corroborated the genetical results. Moreover, the double mutant Macho/deficiens shows only carpels and plena/deficiens only sepals, which is in accord with combinatorial models for homeotic flower formation presented recently.     

The transposable element Tam1 of Antirrhinum majus is 17 kb long

Summary The nivea locus of Antirrhinum majus encodes chalcone synthase, a key enzyme in anthocyanin biosynthesis. Genetic instability is known to occur at this locus. In vitro cloning and characterization of genomic DNA fragments from the unstable nivea-recurrens allele T53 revealed that the instability of nivea-recurrens is due to the presence of a 17 kb DNA insert in the nivea locus. Somatic instability leading to the variegated phenotype, i.e. highly spotted flowers, is due to frequent excision of the 17 kb Tam1 element (Transposon antirrhinum majus) during development of the plant. Excision of Tam1 is not tissue specific, but occurs with similar frequencies in leaves and blossoms.     

Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) 1. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P₇₀₀ upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded Hinf I fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.     

金鱼草中央细胞中线粒体DNA拷贝数的定量

为探究金鱼草(Antirrhinum majus)中央细胞的线粒体DNA拷贝数,采用竞争型定量PCR技术进行了测定。结果表明,金鱼草中央细胞的体积为(61570±732)μm~3,平均携带(783±25)个拷贝的线粒体DNA,并且中央细胞与卵细胞具有相似的体积/线粒体DNA拷贝数比值。推测金鱼草中央细胞包含如此高丰度线粒体DNA可能是为早期胚乳细胞的发育而储备的。     

Rates of movement of transposable elements inDrosophila melanogaster

Mobilization rates of nine families of transposable elements (P, hobo, FB, gypsy, 412, copia, blood, 297, andjockey) were estimated by using 182 lines. Lines were started from a completely isogenic population ofDrosophila melanogaster, carrying the markersepia as an indicator of possible contamination, and have been accumulating spontaneous mutations independently for 80 generations of brother-sister (or two double-first-cousin) matings. Transposable element movements have been analyzed in complete genomes by the Southern technique. Mobilization was a rare event, with an average rate of 10^–5 per site per generation. The most active element wasFB. In contrast, the retroelementsgypsy andblood did not move at all. Most changes in restriction patterns were consistent with rearrangements rather than with true transposition. The euchromatic or heterochromatic location of elements was tested by comparing insertion patterns from adults and salivary glands. Certain putative rearrangements involved heterochromatic copies of the retroelements412, copia or297. Clustering of movement across families was observed, suggesting that movement of different families may be non-independent. An association between modified insertion patterns and mutant effects on quantitative traits shows that spontaneous transposition events cause continuous variation.     

The role of subterminal sites of transposable element Ds of Zea mays in excision

Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3 end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.     

Studies on the Mx transposable element system in maize recovered from X-irradiated stocks

Summary The unstable mutant bz-x3m arose in a plant subjected to X-irradiation. The element at the bronze locus is non-autonomous and recombination data indicate that an autonomous element is tightly linked. The autonomous element has been designated Mx (mobile element induced by X-rays) and the non-autonomous element, rMx (responder to Mx). Linkage data indicate that a second Mx lies near the end of the short arm of chromosome 9; in one plant, an Mx that is unlinked was detected. Distinguishing characteristics of bz-x3m are a large window of time in endosperm development during which somatic reversions can arise and a wide range in the frequency at which they occur; these features are heritable. With increasing doses of bz-x3m and Mx, the window expands and the frequency range increases. In kernels containing the bz-x3m allele and the tightly linked Mx, breakage occurs in chromosome 9 distal to the C locus, resulting in breakage-fusion-bridge patterns for endosperm markers that lie proximal to the break. The frequency of breaks and the developmental time at which they occur exhibit the same dosage effect as the somatic reversions of the bz-x3m allele. These observations suggest that an rMx (designated rMxBr) that causes chromosome breakage is positioned distal to the C locus. At the molecular level, the bz-x3m allele is associated with a 0.5 kb increase in fragment size in DNA samples digested with BglII, EcoRI, HindIII and PstI; in germinal revertants, the fragment size returns to that of the progenitor.     

Molecular analysis of theBg-rbg transposable element system ofZea mays L.

Summary The two components of theBg-rbg transposable element system of maize have been cloned. TheBg element, isolated from the mutable allelewx-m32 :: Bg is inserted in the intron of theWaxy (Wx) gene between exons 12 and 13. The length of the element is of 4869 bp.Bg has 5 by terminal inverted repeats, and generates upon insertion an 8 by direct duplication of the target sequence. Both ends of theBg element contain a 76 by direct repeat adjacent to the terminal inverted repeats. The hexamer motif TATCG_kC ^G is here repeated several times in direct or inverse orientation. Therbg element was isolated from the mutable alleleo2m(r) where it is located in the promoter region of theOpaque-2 (O2) gene.rbg is approximately 4.5 kb in length, has terminal inverted repeats identical to those of theBg element, and is also flanked by an 8 by direct duplication at the target site. LikeBg, rbg carries the 76 by direct repeats. Restriction enzyme analysis reveals that, compared toBg, the receptor element is distinguishable by small deletion and insertion events. Sequence data indicate that not more than 75% homology exists at the DNA level between therbg element and the autonomousBg element.     

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     

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. Received: 22 April 1998 / Accepted: 15 June 1998     

Characterization ofGandalf, a new inverted-repeat transposable element ofDrosophila koepferae

The cloning and characterization ofGandalf, a new DNA-transposing mobile element obtained from theDrosophila koepferae (repleta group) genome is described. A fragment ofGandalf was found in a middle repetitive clone that shows variable chromosomal localization. Restriction, Southern blot, PCR and sequencing analyses have shown that mostGandalf copies are about 1 kb long, are flanked by 12 by inverted terminal repeats and contain subterminal repetitive regions on both sides of the element. As with other elements of the DNA-transposing type (known as the Ac family), theGandalf element generates 8 by direct duplications at the insertion point. Coding region analysis has shown that the longer open reading frame found inGandalf copies could encode part of a protein. However, whether or not the 1 kb copies of the element are actually the active transposons remains to be elucidated.Gandalf shows a very low copy number inD. buzzatii, a sibling species ofD. koepferae. An attempt to induce interspecific hybrid dysgenesis in hybrids of these two species has been unsuccessful.     

Mu transposable elements are structurally diverse and distributed throughout the genusZea

Summary The Robertson's Mutator stock of maize exhibits a high mutation rate due to the transposition of theMu family of transposable elements. All characterizedMu elements contain similar 200-bp terminal inverted repeats, yet the internal sequences of the elements may be completely unrelated. Non-Mutator stocks of maize have a 20–100-fold lower mutation rate relative to Mutator stocks, yet they contain multiple sequences that hybridize to theMu terminal inverted repeats. Most of these sequences do not cohybridize to internal regions of previously clonedMu elements. We have cloned two such sequences from the maize line B37, a non-Mutator inbred line. These sequences, termedMu4 andMu5, have an organization characteristic of transposable elements and possess 200-bpMu terminal inverted repeats that flank internal DNA, which is unrelated to other clonedMu elements.Mu4 andMu5 are both flanked by 9-bp direct repeats as has been observed for otherMu elements. However, we have no direct evidence that they have recently transposed because they have not been found in known genes. Although the internal regions ofMu4 andMu5 are not related by sequence similarity, both elements share an unusual structural feature: the terminal inverted repeats extend more than 100 bp internally fromMu-similar termini. The distribution of these elements in maize lines and related species suggests thatMu elements are an ancient component of the maize genome. Moreover, the structure of theMu termini and the fact thatMu termini are found flanking different internal sequences leads us to speculate thatMu termini once may have been capable of transposing as independent entities.     

Identification and structural characterization of further DNA elements in the potato and pepper genomes homologous to the transposable element-like insertion Tst1

Summary The molecular cloning and nucleotide sequence of elements from potato and pepper that are related to the recently identified Tst1 element are described. Sequence analysis reveals considerable conservation of sequences internal to both the Tst1 element and two of the related elements identified here. In six potato clones analysed, the II by inverted repeat first identified in the Tst1 element is conserved. Several of the elements are flanked by an 8 by direct repeat. DNA fragments which were amplified from several pepper genomes by polymerase chain reaction (PCR) amplification using the inverted repeat as sequence primers also display considerable conservation of sequences internal to the Tst1 element. These data further support the possibility that Tst1 is a non-autonomous transposable element and that Tst1 might be the first example of a transposable element which occurs in several genera of solanaceous plants.     

Chromosome labeling with transposable elements in maize

Transposable elements randomly insert into a targeted locus at a frequency of 10^-6 to 10^-5. The En element has been shown in previous studies to transpose more frequently into closely linked sites. Thus, it is appropriate to place an En element onto each of the 20 chromosome arms in maize to maximize tagging efficiency. This is called chromosome labeling for tagging purposes with transposons. After a chromosome arm has been labeled with a transposon, genes residing in that arm will have a greater chance to be tagged by the transposon. To date, all of the maize chromosome arms have been labeled with at least one of five Encontaining alleles. The elements were linked to the arms using reciprocal translocations. The usage of these arm-labeled lines is discussed in the context of gene tagging.Journal Paper No. 15224 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa; Project No. 3176