Mobile elements and transposition events in the cut locus of Drosophila melanogaster

We have cloned from the Oregon R strain of Drosophila melanogaster a 240 kb segment of DNA that contains the cut (ct) locus, and characterized the region for the presence of repetitive elements. Within this region at least five copies of the suffix element were detected, as well as several putatively novel mobile elements. A number of mutations obtained from the unstable ct ^MR2 strain and its derivatives were mapped within the cut locus. Comparison between parental and daughter strains indicates that frequently two or more independent transposition events involving the cut locus occur simultaneously within a single germ cell, thus providing a molecular basis for the transposition explosion phenomenon.     

The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.     

Genome-Wide Analysis of the Fusarium oxysporum mimp Family of MITEs and Mobilization of Both Native and De Novo Created mimps

We have performed a genome-wide analysis of the mimp family of miniature inverted-repeat transposable elements, taking advantage of the recent release of the F. oxysporum genome sequence. Using different approaches, we detected 103 mimp elements, corresponding to 75 nonredundant copies, half of which are located on a single small chromosome. Phylogenetic analysis identified at least six subfamilies, all remarkably homogeneous in size and sequence. Based on high sequence identity in the terminal inverted repeats (TIRs), mimp elements were connected to different impala members. To gain insights into the mechanisms at the origin and amplification of mimps, we studied the potential of impala to cross-mobilize different mimps, native but also created de novo by inserting a short DNA segment between two TIRs. Our results show that TIR sequences are the main requirement for mobilization but that additional parameters in the internal region are likely to influence transposition efficiency. Finally, we show that integration site preference of native versus newly transposed mimps greatly varies in the host genomes used in this study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequences of novel mimp3 and mimp4 elements are available under GenBank accession numbers EU833100 and EU833101, respectively. Coordinates of mimp5, mimp6 and of non-classified mimp copies are indicated in Supplementary Table 1.     

A small sequence in domain v of the mitochondrial large ribosomal RNA restores <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> pole cell determination in uv-irradiated embryos

The mechanism by which the mitochondrial large rRNA is involved in the restoration of the pole cell-forming ability in Drosophila embryos is still unknown. We identified a 15-ribonucleotide sequence which is conserved from the protobacterium Wolbachia to the higher eukaryotes in domain V of the mitochondrial large rRNA. This short sequence is sufficient to restore pole cell determination in UV-irradiated Drosophila embryos. Here, we provide evidence that the conserved 15-base sequence is sufficient to restore luciferase activity in vitro. Moreover, we show that the internal GAGA sequence is involved in protein binding and that mutations in this tetranucleotide affect the sequence’s ability to restore luciferase activity. The obtained results lead us to propose that mtlrRNA may be involved either in damaged protein reactivation or in protein biosynthesis during pole cell determination.     

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.     

Three highly divergent subfamilies of the impala transposable element coexist in the genome of the fungus Fusarium oxysporum

The transposable element impala is a member of the widespread superfamily of Tc1-mariner transposons, identified in the genome of the plant pathogenic fungus Fusarium oxysporum. This element is present in a low copy number and is actively transposed in the F.␣oxysporum strain F24 that is pathogenic for melons. The structure of the impala family was investigated by cloning and sequencing all the genomic copies. The analysis revealed that this family is composed of full-length and truncated copies. Four copies contained a long open reading frame that could potentially encode a transposase of 340 amino acids. The presence of conserved functional domains (a nuclear localisation signal, a catalytic DDE domain and a DNA-binding domain) suggests that these four copies may be autonomous elements. Sequence comparisons and phylogenetic analysis of the impala copies defined three subfamilies, which differ by a high level of nucleotide polymorphism (around 20%). The coexistence of these divergent subfamilies in the same genome may indicate that the impala family is of ancient origin and/or that it arose by successive horizontal transmission events. Received: 2 December 1997 / Accepted: 28 April 1998     

Maize Activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats

 The mobility of maize transposable element Activator (Ac) is dependent on the 11-bp terminal inverted repeats (IRs) and approximately 250 subterminal nucleotides at each end. These sequences flank the coding region for the transposase (TPase) protein, which is required for the transposition reaction. Here we show that Ac TPase has a bipartite DNA binding domain, and recognizes the IRs and subterminal sequences in the Ac ends. TPase binds cooperatively to repetitive ACG and TCG sequences, of which 25 copies are found in the 5′ and 20 copies in the 3′ subterminal regions. TPase affinity is highest when these sites are flanked on the 3′ side by an additional G residue (A/TCGG), which is found at 75% of binding sites. Moreover, TPase binds specifically to the Ac IRs, albeit with much lower affinity. Two mutations within the IRs that immobilize Ac abolish TPase binding completely. The basic DNA binding domain of TPase is split into two subdomains. Binding to the subterminal motifs is accomplished by the C-terminal subdomain alone, whereas recognition of the IRs requires the N-terminal subdomain in addition. Furthermore, TPase is extremely flexible in DNA binding. Two direct or inverted binding sites are bound equally well, and sites that are five to twelve bases apart are similarly well bound. The consequences of these findings for the Ac transposition reaction are discussed. Received: 3 June 1996 / Accepted: 29 July 1996     

A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function.     

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3 end of the element on germinal excision frequencies. Alteration of three bases to create a BglII site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.     

Chromosomal polymorphism of ribosomal genes in the genus Oryza

The genes encoding for 18S–5.8S–28S ribosomal RNA (rDNA) are both conserved and diversified. We used rDNA as probe in the fluorescent in situ hybridization (rDNA-FISH) to localized rDNAs on chromosomes of 15 accessions representing ten Oryza species. These included cultivated and wild species of rice, and four of them are tetraploids. Our results reveal polymorphism in the number of rDNA loci, in the number of rDNA repeats, and in their chromosomal positions among Oryza species. The numbers of rDNA loci varies from one to eight among Oryza species. The rDNA locus located at the end of the short arm of chromosome 9 is conserved among the genus Oryza. The rDNA locus at the end of the short arm of chromosome 10 was lost in some of the accessions. In this study, we report two genome specific rDNA loci in the genus Oryza. One is specific to the BB genome, which was localized at the end of the short arm of chromosome 4. Another may be specific to the CC genome, which was localized in the proximal region of the short arm of chromosome 5. A particular rDNA locus was detected as stretched chromatin with bright signals at the proximal region of the short arm of chromosome 4 in O. grandiglumis by rDNA-FISH. We suggest that chromosomal inversion and the amplification and transposition of rDNA might occur during Oryza species evolution. The possible mechanisms of cyto-evolution in tetraploid Oryza species are discussed.     

IS30, a new insertion sequence of Escherichia coli K12

Summary Three independent spontaneous mutations of prophage P1 affecting the ability of the phage to reproduce vegetatively are due to the insertion of a mobile genetic element, called IS 30. The same sequence is also carried in the R plasmid NR 1-Basel, but not in the parental plasmid NR 1. Southern hybridisation study indicates that the Escherichia coli K 12 chromosome carries several copies of IS 30 as a normal resident. IS 30 is 1.2 kb long and contains unique restriction cleavage sites for Bg/II, ClaI, HindIII, NciI and HincII, and it is cleaved twice by the enzymes HpaII and TaqI. The ends of IS 30 are formed by 26 bp long inverted repeats with 3 bases mismatched. Upon transposition IS 30 generates a duplication of only 2 bp of the target. The following observations suggest a pronounced specificity in target selection by IS 30. In transposition to the phage P 1 genome a single integration site was used three times independently, and in both orientations. A short region of sequence homology has been identified between the P 1 and NR 1-Basel insertion sites. IS 30 has mediated cointegration as well as deletion. The entire IS 30 sequences were duplicated in the cointegrates between a pBR 322 derivative containing IS 30 and the genome of phage P 1–15, and several loci on the P1–15 genome served as fusion sites, some of which were used more than once.     

Extensive Amplification and Transposition of a Novel Repetitive Element, Xstir, Together with Its Terminal Inverted Repeat in the Evolution of Xenopus

A DNA fragment containing short tandem repeat sequences (approximately 86-bp repeat) was isolated from a Xenopus laevis cDNA library. Southern blot and in situ hybridization analyses revealed that the repeat was highly dispersed in the genome and was present at approximately 1 million copies per haploid genome. We named this element Xstir (Xenopus short tandemly and invertedly repeating element) after its arrangement in the genome. The majority of the genomic Xstir sequences were digested to monomer and dimer sizes with several restriction enzymes. Their sequences were found to be highly homogeneous and organized into tandem arrays in the genome. Alignment analyses of several known sequences showed that some of the Xstir-like sequences were also organized into interspersed inverted repeats. The inverted repeats consisted of an inverted pair of two differently modified Xstirs separated by a short insert. In addition, these were framed by another novel inverted repeat (Xstir-TIR). The Xstir-TIR sequence was also found at the ends of tandem Xstir arrays. Furthermore, we found that Xstir-TIR was linked to a motif characterizing the T2 family which belonged to a vertebrate MITE (miniature inverted-repeat transposable element) family, suggesting the importance of Xstir-TIR for their amplification and transposition. The present study of 11 anuran and 2 urodele species revealed that Xstir or Xstir-like sequences were extensively amplified in the three Xenopus species. Genomic Xstir populations of X. borealis and X. laevis were mutually indistinguishable but significantly different from that of X. tropicalis. Received: 5 April 2000 / Accepted: 3 August 2000     

Genealogy of the α-crystallin—small heat-shock protein superfamily

Sequences of 40 very diverse representatives of the α-crystallin–small heat-shock protein (α-Hsp) superfamily are compared. Their characteristic C-terminal ‘α-crystallin domain' of 80–100 residues contains short consensus sequences that are highly conserved from prokaryotes to eukaryotes. There are, in addition, some positions that clearly distinguish animal from non-animal α-Hsps. The α-crystallin domain is predicted to consist of two hydrophobic β-sheet motifs, separated by a hydrophilic region which is variable in length. Combination of a conserved α-crystallin domain with a variable N-terminal domain and C-terminal extension probably modulates the properties of the various α-Hsps as stress-protective and structural oligomeric proteins. Phylogeny reconstruction indicates that multiple α-Hsps were already present in the last common ancestor of pro- and eukaryotes. It is suggested that during eukaryote evolution, animal and non-animal α-Hsps originated from different ancestral gene copies. Repeated gene duplications gave rise to the multiple α-Hsps present in most organisms.     

Structure and function of repetitive sequence elements associated with a highly polymorphic domain of the Neisseria meningitidis PilQ protein

Secretins are a large family of proteins associated with membrane translocation of macromolecular complexes, and a subset of this family, termed PilQ proteins, is required for type IV pilus biogenesis. We analysed the status of PilQ expression in Neisseria meningitidis (Mc) and found that PilQ^? mutants were non-piliated and deficient in the expression of pilus-associated phenotypes. Sequence analysis of the 5′ portion of the pilQ ORF of the serogroup B Mc strain 44/76 showed the presence of seven copies of a repetitive sequence element, in contrast to the situation in N. gonorrhoeae (Gc) strains, which carry either two or three copies of the repeat. The derived amino acid sequence of the consensus nucleotide repeat was an octapeptide PAKQQAAA, designated as the small basic repeat (SBR). This gene segment was studied in more detail in a collection of 52 Mc strains of diverse origin by screening for variability in the size of the PCR-generated DNA fragments spanning the SBRs. These strains were found to harbour from four to seven copies of the repetitive element. No association between the number of copies and the serogroup, geographic origin or multilocus genotype of the strains was evident. The presence of polymorphic repeat elements in Mc PilQ is unprecedented within the secretin family. To address the potential function of the repeat containing domain, Mc strains were constructed so as to express chimeric PilQ molecules in which the number of SBR repeats was increased or in which the repeat containing domain was replaced in toto by the corresponding region of the Pseudomonas aeruginosa (Pa) PilQ protein. Although the strain expressing PilQ with an increased number of SBRs was identical to the parent strain in pilus phenotypes, a strain expressing PilQ with the equivalent Pa domain had an eightfold reduction in pilus expression level. The findings suggest that the repeat containing domain of PilQ influences Mc pilus expression quantitatively but not qualitatively.     

Analysis of re-integrated Ac element positions in the genome of Populus provides a basis for Ac/Ds-transposon activation tagging in trees

With a view to establish an efficient gene tagging system for forest tree species, we assessed the transposition behaviour of the maize transposable element Ac in poplar. In earlier work, we showed that new integration sites were often located within predicted or known coding sequences. However, somatic transposition behaviour of Ac with regard to conserved chromosome specificity or, more specifically, whether Ac transposition is restricted to the chromosome on which the primary insertion locus (donor) is located or whether it is able to pass chromosomal boundaries, remained unclear. To answer these questions, we took advantage of the publicly available Populus trichocarpa genome sequence (Phytozome v5.0; ) and three 35S::Ac-rolC transgenic hybrid aspen lines to determine the flanking sequences of Ac re-integration sites for tissue sectors from which Ac had been excised. Only about one-third of the analysed re-integrations were positioned within the scaffold containing the primary Ac donor locus, and the majority of re-integrations were found scattered over many unlinked sites on other scaffolds confirming that Ac transposition in poplar does in fact cross chromosome boundaries. The majority of re-integration sites (57.1%) were found within or near coding regions demonstrating that Ac/Ds transposon tagging in poplar holds much promise for the efficient induction of mutants and functional genomics studies in forest tree species.