Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

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.     

Two distinct P element subfamilies in the genome of Drosophila bifasciata

The genome of Drosophila bifasciata harbours two distinct subfamilies of P-homologous sequences, designated M-type and O-type elements based on similarities to P element sequences from other species. Both subfamilies have some general features in common: they are of similar length (M-type: 2935 bp, O-type: 2986 bp), are flanked by direct repeats of 8 by (the presumptive target sequence), contain terminal inverted repeats, and have a coding region consisting of four exons. The splice sites are at homologous positions and the exons have the coding capacity for proteins of 753 amino acids (M-type) and 757 amino acids (O-type). It seems likely that both types of element represent functional transposons. The nucleotide divergence of the two P element subfamilies is high (31%). The main structural difference is observed in the terminal inverted repeats. Whereas the termini of M-type elements consist of 31 by inverted repeats, the inverted repeats of the O-type elements are interrupted by non-complementary stretches of DNA, 12 by at the 5 end and 14 by at the 3 end. This peculiarity is shared by all members of the O-type subfamily. Comparison with other P element sequences indicates incongruities between the phylogenies of the species and the P transposons. M-type and O-type elements apparently have no common origin in the D. bifasciata lineage. The M-type sequence seems to be most closely related to the P element from Scaptomyza pallida and thus could be considered as a more recent invader of the D. bifasciata gene pool. The origin of the O-type elements cannot be unequivocally deduced from the present data. The sequence comparison also provides new insights into conserved domains with possible implications for the function of P transposons.     

Identification of Tn4430, a transposon of Bacillus thuringiensis functional in Escherichia coli

Summary The mobile genetic element Tn4430, originating from the gram-positive bacterium, Bacillus thuringiensis, and previously described as the Th-sequence, is the first transposon isolated from the genus Bacillus. In the present work a gene (APH-III) conferring resistance to kanamycin was inserted into this 4.2 kb transposon. Transposition experiments showed that Tn4430APH-III could transpose in the gram-negative host Escherichia coli when its insertion functions were supplied by an intact copy of Tn4430. By transposing Tn4430APH-III directly onto pBR322, it was possible to determine the nucleotide sequence of the terminal inverted repeats of Tn4430 and of the target DNA site. Identical 38 bp in inverted orientation are situated at each end of the transposon and there is a direct duplication of 5 bp at the insertion site. Thus, it is clear that Tn4430 is closely related to the transposons belonging to the Tn3 family (class II elements).     

Isolation and molecular characterization of dTnp1, a mobile and defective transposable element of Nicotiana plumbaginifolia

By Northern blot analysis of nitrate reductase-deficient mutants of Nicotiana plumbaginifolia, we identified a mutant (mutant D65), obtained after -ray irradiation of protoplasts, which contained an insertion sequence in the nitrate reductase (NR) mRNA. This insertion sequence was localized by polymerase chain reaction (PCR) in the first exon of NR and was also shown to be present in the NR gene. The mutant gene contained a 565 by insertion sequence that exhibits the sequence characteristics of a transposable element, which was thus named dTnp1. The dTnp1 element has 14 by terminal inverted repeats and is flanked by an 8-bp target site duplication generated upon transposition. These inverted repeats have significant sequence homology with those of other transposable elements. Judging by its size and the absence of a long open reading frame, dTnp1 appears to represent a defective, although mobile, transposable element. The octamer motif TTTAGGCC was found several times in direct orientation near the 5 and 3 ends of dTnp1 together with a perfect palindrome located after the 5 inverted repeat. Southern blot analysis using an internal probe of dTnp1 suggested that this element occurs as a single copy in the genome of N. plumbaginifolia. It is also present in N. tabacum, but absent in tomato or petunia. The dTnp1 element is therefore of potential use for gene tagging in Nicotiana species.     

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.     

A candidate autonomous version of the wheat MITE <Emphasis Type="Italic">Hikkoshi</Emphasis> is present in the rice genome

A miniature inverted-repeat transposable element (MITE), designated as Hikkoshi, was previously identified in the null Wx-A1 allele of Turkish bread wheat lines. This MITE is 165 bp in size and has 12-bp terminal inverted repeats (TIRs) flanked by 8-bp target site duplications (TSDs). Southern and PCR analyses demonstrated the presence of multiple copies of Hikkoshi in the wheat genome. Database searches indicated that Hikkoshi MITEs are also present in barley, rice and maize. A 3.4-kb element that has Hikkoshi-like TIRs flanked by 8-bp TSDs has now been identified in the rice genome. This element shows high similarity to the 5 subterminal region of the wheat Hikkoshi MITE and contains a transposase (TPase) coding region. The TPase has two conserved domains, ZnF_TTF and hATC, and its amino acid sequence shows a high degree of homology to TPases encoded by Tip100 transposable elements belonging to the hAT superfamily. We designated the 3.4-kb element as OsHikkoshi. Several wheat clones deposited in EST databases showed sequence similarity to the TPase ORF of OsHikkoshi. The sequence information from the TPase of OsHikkoshi will thus be useful in isolating the autonomous element of the Hikkoshi system from wheat.     

The isolation of Ant1, a transposable element from Aspergillus niger

A transposable element has been isolated from the industrially important fungus Aspergillus niger (strain N402). The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. It had inserted at a TA site and appeared to have duplicated the target site upon insertion. The isolated element was found to be 4798 by in length and contained 37-bp inverted, imperfect, terminal repeats (ITRs). The sequence of the central region of the element revealed an open reading frame (designated ORF1) which showed similarity, at the amino acid level, to the transposase of the Tc1/mariner class of DNA transposons. Another sequence within the central region of the element showed similarity to the 3 coding and downstream untranslated region of the amyA gene of A. niger. Sequence homology and structural features indicate that this element, which has been named Ant1 (A. niger transposon 1), is related to the Tc1/mariner group of DNA transposons. Ant1 is apparently present as a single copy in strain N402 of A. niger.     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5-CACTA--- -3; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.     

Linear mitochondrial DNAs from yeasts: telomeres with large tandem repetitions

The terminal structure of the linear mitochondrial DNA (mtDNA) from the yeast Candida parapsilosis was investigated. This mtDNA, 30 kb long, has symmetrical ends forming inverted terminal repeats. These repeats are made up of a variable number of tandemly repeating units of 738 by each; the terminal nucleotide corresponds to a precise position within the last repeat unit sequence. The ends had an open structure accessible to enzymes, with a 5 single-stranded extension of about 110 nucleotides. No circular forms were detected in the DNA preparations. Two other unrelated species, Pichia philodendra and Candida salmanticensis also appear to have a linear mtDNA of similar organization. These linear DNAs (which we name Type 2 linear mtDNAs) are distinct from the previously described linear mtDNAs of yeasts whose termini are formed by a closed hairpin loop (Type 1 linear mtDNA). The terminal structure of C. parapsilosis mtDNA is reminiscent of the linear mitochondrial genomes of the ciliate Tetrahymena although, in the latter, the telomeric tandem repeat unit is considerably shorter.     

Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.)

Members of a novel Master family of class II transposons were identified in the carrot genome. Two elements, 2.5 kb long DcMaster1 and 4.4 kb long DcMaster-a, are characterized by 22 bp imperfect terminal inverted repeats and by 3 bp target site duplications. GenBank search revealed that related elements are also present in Medicago truncatula, including a 5.1 kb element MtMaster-a. Both DcMaster-a and MtMaster-a contain open reading frames encoding for putative transposases with the complete DDE domain typical for plant class II transposable elements belonging to PIF/Harbinger superfamily, where the Master elements form a distinct group. Less than 10 copies of the DcMaster element containing the DDE domain are present in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in 8 of 14 identified insertion sites. PCR amplification of carrot genomic DNA using a primer complementary to TIRs of DcMaster gave products <400 bp in size. We speculate that these may all represent a MITE-like family of transposable elements that we named Krak, present in the carrot genome in at least 3,600 copies. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession numbers DQ250792 to DQ250807 and DQ353734 to DQ353752.     

Germinal and somatic products of Mu1 excision from the Bronze-1 gene of Zea mays

Summary Germinal and somatic excision products of Mu1 from the insertion allele bz::mu1 were selectively amplified from maize cob tissue. The sequence of these footprints often included deletions at the target site, suggesting that substantial exonucleolytic degradation occurs upon excision of the element. In addition to deletions of target site sequences, single base insertions were also found. The isolation of an excision product including a 4 by inverted duplication of the target site provides evidence that the double-stranded chromosomal break generated by Mu excision may be terminated by a covalently closed hairpin structure. The majority of excision products, however, do not include inverted duplications of target site sequences, suggesting that such structures are the result of occasional repair activities, rather than an essential step in the mechanism of Mu excision. The sequence of the Mu insertion sites of the bz::mu1 and bz::mu2 alleles is also presented.     

A transposon-like structure in the 5'' flanking sequence of a legumin gene from Pisum sativum

Summary The legumin storage proteins of Pisum sativum are coded for by a multigene family. An insertion element (Pis1) has been found integrated into the 5 flanking sequence of the legC legumin seed storage protein gene. This element contains all the sequence features of the CACTA family of insertion elements, has perfect 12 bp inverted repeats at its termini, and generates a target host site duplication upon integration. An 8 bp sequence within the left arm of the insertion element shows perfect homology to a sequence in the legC flanking region. Three stem-loop structures which can be formed within the element have the same stem sequence.     

Tnr8, a foldback transposable element from rice

An insertion sequence 418 bp in length was found in one member of rice retroposon p-SINE1 in Oryza glaberrima. This sequence had long terminal inverted repeats (TIRs) and is flanked by direct repeats of a 9-bp sequence at the target site, indicative that the insertion sequence is a rice transposable element, which we named Tnr8. Interestingly, each TIR sequence consisted of a unique 9-bp terminal sequence and six tandem repeats of a sequence about 30 bp in length, like the foldback transposable element first identified in Drosophila. A homology search of databases and analysis by PCR revealed that a large number of Tnr8 members with sequence variations were present in the rice genome. Some of these members were not present at given loci in several rice species with the AA genome. These findings suggest that the Tnr8 family members transposed long ago, but some appear to have mobilized after rice strains with the AA genome diverged. The Tnr8 members are thought to be involved in rearrangements of the rice genome.     

Structural and functional analysis of a new retrotransposon class in Drosophila species

Several copies of the Penelope transposable element, previously described in Drosophila virilis, have been studied in different D. virilis strains and D. melanogaster strains transformed with P-based constructs bearing a full-size Penelope copy. Most Penelope copies in both species have large terminal inverted repeats (TIRs) and deletions of various sizes at the 5′ ends of their ORFs. Junctions between TIRs and ORFs usually have microhomologies of various lengths, which allowed a hypothesis explaining the emergence of these complex structures at the molecular level to be put forward. Most Penelope copies have a 34 bp long direct repeat at the ORF ends. Full-size and truncated Penelope copies are usually surrounded by target site duplications of various lengths.     

Whole genome resequencing reveals natural target site preferences of transposable elements in Drosophila melanogaster

Transposable elements are mobile DNA sequences that integrate into host genomes using diverse mechanisms with varying degrees of target site specificity. While the target site preferences of some engineered transposable elements are well studied, the natural target preferences of most transposable elements are poorly characterized. Using population genomic resequencing data from 166 strains of Drosophila melanogaster, we identified over 8,000 new insertion sites not present in the reference genome sequence that we used to decode the natural target preferences of 22 families of transposable element in this species. We found that terminal inverted repeat transposon and long terminal repeat retrotransposon families present clade-specific target site duplications and target site sequence motifs. Additionally, we found that the sequence motifs at transposable element target sites are always palindromes that extend beyond the target site duplication. Our results demonstrate the utility of population genomics data for high-throughput inference of transposable element targeting preferences in the wild and establish general rules for terminal inverted repeat transposon and long terminal repeat retrotransposon target site selection in eukaryotic genomes.     

Fot1, a new family of fungal transposable elements

Summary We report here the discovery of a family of transposable elements, which we refer to as Fotl elements, in the fungal plant pathogen Fusarium oxysporum. The first element was identified as an insertion in the gene encoding nitrate reductase. It is 1928 by long, has 44 by inverted terminal repeats, contains a large open reading frame and is flanked by a 2 by (TA) target site duplication. This element shares significant structural similarities with a class of transposons that includes Tc1 from Caenorhabditis elegans and therefore represents a new class of transposable elements in fungi.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.     

The Tc2 transposon of Caenorhabditis elegans has the structure of a self-regulated element.

We have analyzed the sequence of the Tc2 transposon of the nematode Caenorhabditis elegans. The Tc2 element is 2,074 bp in length and has perfect inverted terminal repeats of 24 bp. The structure of this element suggests that it may have the capacity to code for a transposase protein and/or for regulatory functions. Three large reading frames on one strand exhibit nonrandom codon usage and may represent exons. The first open coding region is preceded by a potential CAAT box, TATA box, and consensus heat shock sequence. In addition to its inverted terminal repeats, Tc2 has an unusual structural feature: subterminal degenerate direct repeats that are arranged in an irregular overlapping pattern. We have also examined the insertion sites of two Tc2 elements previously identified as the cause of restriction fragment length polymorphisms. Both insertions generated a target site duplication of 2 bp. One element had inserted inside the inverted terminal repeat of another transposon, splitting it into two unequal parts.