Distinct characteristics of loop sequences of two Drosophila foldback transposable elements.

A few foldback (FB) transposable elements have, between their long terminal inverted repeats, central loop sequences which have been shown to be different from FB inverted repeat sequences. We have investigated loop sequences from two such FB elements by analyzing their genomic distribution and sequence conservation and, in particular, by determining if they are normally associated with FB elements. One of these FB loop sequences seems to be present in a few conserved copies found adjacent to FB inverted repeat sequences, suggesting that it represents an integral component of some FB elements. The other loop sequence is less well-conserved and not usually associated with FB inverted repeats. This sequence is a member of another family of transposable elements, the HB family, and was found inserted in an FB element only by chance. We compare the complete DNA sequences of two HB elements and examine the ends of four HB elements.     

Molecular and bioinformatic analysis of the FB-NOF transposable element

The Drosophila melanogaster transposable element FB-NOF is known to play a role in genome plasticity through the generation of all sort of genomic rearrangements. Moreover, several insertional mutants due to FB mobilizations have been reported. Its structure and sequence, however, have been poorly studied mainly as a consequence of the long, complex and repetitive sequence of FB inverted repeats. This repetitive region is composed of several 154 bp blocks, each with five almost identical repeats. In this paper, we report the sequencing process of 2 kb long FB inverted repeats of a complete FB-NOF element, with high precision and reliability. This achievement has been possible using a new map of the FB repetitive region, which identifies unambiguously each repeat with new features that can be used as landmarks. With this new vision of the element, a list of FB-NOF in the D. melanogaster genomic clones has been done, improving previous works that used only bioinformatic algorithms. The availability of many FB and FB-NOF sequences allowed an analysis of the FB insertion sequences that showed no sequence specificity, but a preference for A/T rich sequences. The position of NOF into FB is also studied, revealing that it is always located after a second repeat in a random block. With the results of this analysis, we propose a model of transposition in which NOF jumps from FB to FB, using an unidentified transposase enzyme that should specifically recognize the second repeat end of the FB blocks.     

FB elements are the common basis for the instability of the wDZL and wC Drosophila mutations

The DNA insertions that cause the highly unstable mutations wC and wDZL share extensive homology with the FB family of transposable elements. FB elements carry long, internally repetitious, inverted terminal repeats and thus differ in structure from other transposable elements. Our results suggest that FB elements may excise and cause chromosomal rearrangements at unusually high frequencies. The wC insertion is a single FB element. The wDZL insertion differs in that it contains two FB elements, one at each terminus. The wC and wDZL insertions contain 4.0 and 6.5 kilobase nonhomologous segments between their terminal repeats. In contrast to the middle repetitive FB elements, the central segment of the wDZL insertion is single-copy and present at a fixed location in the wild-type genome. It has apparently been transposed by the action of flanking FB elements, causing the wDZL mutation at its new location.     

Complete foldback transposable elements encode a novel protein found in Drosophila melanogaster.

An apparently complete foldback (FB) transposable element homologous to FB white-crimson (FBwc) was analyzed. A complete FB element could encode one or more proteins required for regulation of FB transposition. The central DNA region (the loop) and the junctions between the loop and the inverted terminal repeats were sequenced. Three open reading frames (ORFs) are present in the loop, and a novel 308 bp inverted repeat is present at the junctions. No significant homologies were found when the DNA sequences of the loop region and the novel inverted repeat were screened against the Gene data bank. Antibodies were prepared in guinea-pigs against a peptide present near the amino terminus of ORF1, the longest ORF. A 71,000 dalton protein was isolated from an extract of Drosophila melanogaster early-stage embryos on an anti-ORF1 peptide-affinity column. Immunohistochemical studies of adult flies demonstrate localization of this protein in egg chambers.     

MMTS, a new subfamily of Tc1-like transposons

A novel Tc1-like transposable element has been identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. The M. mizolepis Tc1-like transposon (MMTS) is comprised of inverted terminal repeats and a single gene that codes Tc1-like transposase. The deduced amino acid sequence of the transposase-encoding region of MMTS transposon contains motifs including DDE motif, which was previously recognized in other Tc1-like transposons. However, putative MMTS transposase has only 34-37% identity with well-known Tc1, PPTN, and S elements at the amino acid level. In dot-hybridization analysis used to measure the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that the MMTS transposon is present at about 3.36 x 104 copies per 2 x 109 bp, and accounts for approximately 0.027% of the mud loach genome. Here, we also describe novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fishes, which bear conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches and phylogenetic analysis indicated that MMTS-like transposons evolved uniquely in fishes, and comprise a new subfamily of Tc1-like transposons, with only modest similarity to Drosophila melanogaster (foldback element FB4, HB2, HB1), Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae (Frisky).     

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.     

The inverted repeats of IS<Emphasis Type="Italic">1384</Emphasis>, a newly described insertion sequence from<Emphasis Type="Italic"> Pseudomonas putida</Emphasis> strain H,represent the specific target for integration of IS<Emphasis Type="Italic">1383</Emphasis>

Analysis of a region on plasmid pPGH1 from Pseudomonas putida strain H that is flanked by two copies of IS1383 has revealed an additional element with the typical features of a bacterial insertion sequence. This new IS element, designated IS1384, contains a single ORF of 972 bp, and is flanked by 9-bp inverted repeats. Based on sequence homology and structural characteristics of the putative transposase it encodes, IS1384 belongs to the IS5 subgroup of the IS5 family. Two copies of IS1384 are present on plasmid pPGH1, whereas none could be detected on the chromosome of P. putida strain H. Sequence analysis revealed the presence of two truncated copies of IS1384 on the second plasmid in this strain, pPGH2. The inverted repeats of all IS1384 copies (including the truncated ones) are interrupted by the integration of an IS1383 element. All integrations were found to be site- and orientation-specific. PCR studies and sequence data indicate that IS1383 can form a circular intermediate on excision. In the circular form, the previously described 13-bp inverted repeats of IS1383 are separated by 10 bp that are identical to the 5-bp motif that flanks each side of the element when it is integrated in its target. We provide evidence that these additional nucleotides, although not of inverted symmetry, represent an essential part of the inverted repeats. Furthermore, the data indicate that IS1383 integrated into the inverted repeats of IS1384 by a site-specific recombination rather than a site-specific insertion event.     

Developmentally coordinated en masse excision of a highly repetitive element in E. crassus

The E. crassus Tec1 element is present in greater than 10(4) copies in the micronuclear genome but is absent from the macronuclear genome. During formation of a macronucleus from a micronucleus, a majority of the Tec1 elements appear as extrachromosomal circles. The circular and integrated forms of Tec1 have been characterized by restriction mapping to produce consensus maps and by sequence analysis of the element's termini. The circular forms are resistant to BAL31 and have the restriction map expected if the element excises at the end of its inverted repeats. DNA sequence analysis of a circular form confirms that the inverted repeats are in a head-to-head configuration. Excision of Tec1 occurs very early during macronuclear development as the DNA begins to replicate to form polytene chromosomes.     

Characterization of the Fb-Nof Transposable Element of Drosophila Melanogaster

FB-NOF is a composite transposable element of Drosophila melanogaster. It is composed of foldback sequences, of variable length, which flank a 4-kb NOF sequence with 308-bp inverted repeat termini. The NOF sequence could potentially code for a 120-kD polypeptide. The FB-NOF element is responsible for unstable mutations of the white gene (wc and wDZL) and is associated with the large TEs of G. Ising. Although most strains of D. melanogaster have 20-30 sites of FB insertion, FB-NOF elements are usually rare, many strains lack this composite element or have only one copy of it. A few strains, including wDZL and Basc have many (8-21) copies of FB-NOF, and these show a tendency to insert at "hot-spots." These strains also have an increased number of FB elements. The DNA sequence of the NOF region associated with TE146(Z) has been determined.     

Sequence of ISRm4 from Rhizobium meliloti strain GR4.

ISRm4, an IS-like sequence structurally similar to Pseudomonas cepacia insertion element IS402, was identified by sequence analysis. This 933-bp element carries 17-bp putative terminal inverted repeats with five mismatches and a putative direct target duplication of 3 bp.     

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.     

Pogo transposase contains a putative helix-turn-helix DNA binding domain that recognises a 12 bp sequence within the terminal inverted repeats.

Pogo is a transposable element with short terminal inverted repeats. It contains two open reading frames that are joined by splicing and code for the putative pogo transposase, the sequence of which indicates that it is related to the transposases of members of the Tc1/mariner family as well as proteins that have no known transposase activity including the centromere binding protein CENP-B. We have shown that the N-terminal region of pogo transposase binds in a sequence-specific manner to the ends of pogo and have identified residues essential for this. The results are consistent with a prediction that DNA binding is due to a helix-turn-helix motif within this region. The transposase recognises a 12 bp sequence, two copies of which are present at each end of pogo DNA. The outer two copies occur as inverted repeats 14 nucleotides from each end of the element, and contain a single base mismatch and indicate the inverted repeats of pogo are 26 nucleotides long. The inner copies occur as direct repeats, also with a single mismatch.     

A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.

The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.     

The 1723 element: a long, homogeneous, highly repeated DNA unit interspersed in the genome of Xenopus laevis

We describe a highly repeated DNA element in the Xenopus laevis genome. This sequence, named the 1723 element, was first identified among sequences that are transcribed during embryonic development. The element is present in about 8500 copies per haploid genome, which together accounts for about 2.4% of the genome. Most copies of the element have highly conserved restriction maps, and are interspersed in the genome. The copies range in size from 6000 to 10,000 base-pairs due to an expandable region that contains variable numbers of a tandemly repeating 183 to 204 base-pair unit. The element is framed by an imperfect 18 base-pair inverted sequence, and inverted repeats of 180 to 185 base-pairs are nearby. Sequence analysis of DNA adjacent to three cloned elements shows that the elements are flanked by 8 base-pair direct repeats. These and other properties of 1723 suggest that it may be transposable.     

Characterization of IS476 and its role in bacterial spot disease of tomato and pepper.

IS476 is an endogenous insertion sequence present in copper-tolerant strains of Xanthomonas campestris pv. vesicatoria. Sequence analysis has revealed that the element is 1,225 base pairs in length, has 26-base-pair inverted repeats, and causes a 4-base-pair target site duplication upon insertion into the avirulence gene avrBs1. Comparison of the full-length sequence with sequences in the National Biomedical Research Foundation and National Institutes of Health data bases showed that one of the predicted IS476 proteins is partially homologous to the putative transposase of IS3 from Escherichia coli, and the inverted repeats of IS476 have significant homology to the inverted repeats of the IS51 insertion sequence of Pseudomonas syringae pv. savastanoi. A transposition assay based on the insertional inactivation of the sacRB locus of Bacillus subtilis was used to demonstrate that one of the three copies of IS476 residing on the 200-kilobase copper plasmid pXVCU1 is capable of transposition in several strains of Xanthomonas campestris. The position of IS476 insertion in several avrBs1 mutants was established and was shown to influence both induction of hypersensitivity and bacterial growth in planta.     

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.     

Structure and evolution of a family of interspersed repetitive DNA sequences inCaenorhabditis elegans

Summary The structure of three members of a repetitive DNA family from the genome of the nematodeCaenorhabditis elegans has been studied. The three repetitive elements have a similar unitary structure consisting of two 451-bp sequences in inverted orientation separated by 491 bp, 1.5 kb, and 2.5 kb, respectively. The 491-bp sequence separating the inverted 451-bp sequences of the shortest element is found adjacent to one of the repeats in the other two elements as well. The combination of the three sequences we define as the basic repetitive unit. Comparison of the nucleotide sequences of the three elements has allowed the identification of the one most closely resembling the primordial repetitive element. Additionally, a process of co-evolution is evident that results in the introduction of identical sequence changes into both copies of the inverted sequence within a single unit. Possible mechanisms are discussed for the homogenization of these sequences. A direct test of one possible homogenization mechanism, namely homologous recombination between the inverted sequences accompanied by gene conversion, shows that recombination between the inverted repeats does not occur at high frequency.     

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.     

The novel insertion sequences IS1417, IS1418, and IS1419 from Burkholderia glumae and their strain distribution

Three insertion sequences, IS1417, IS1418, and IS1419, were isolated from Burkholderia glumae (formerly Pseudomonas glumae), a gram-negative rice pathogenic bacterium, on the basis of their abilities to activate the expression of the neo gene of the entrap vector pSHI1063. The 1335-bp IS1417 element with 17-bp imperfect terminal inverted repeats was found to be flanked by 5-bp direct repeats of the vector sequence. IS1418 is 865 bp in length and carries 15-bp inverted repeats with a target duplication of 3 bp. The 1215-bp IS1419 sequence is bounded by the 36-bp terminal inverted repeats of the element and 7-bp direct repeats of the vector sequence. IS1417 and IS1418 belong to the IS2 subgroup of the IS3 family and the IS427 subgroup of the IS5 family, respectively, whereas IS1419 does not appear to be a member of any known IS family. Southern blot analysis of DNAs from B. glumae field isolates indicated that those IS elements are widely distributed, but the host range of the three IS elements appears to be limited to B. glumae and some other related species such as B. plantarii. The polymorphisms exhibited in B. glumae isolates suggest that those elements are useful for molecular epidemiological studies of B. glumae infections.