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A new insertion sequence (IS), IS 1642, was identified in a Mycobacterium avium strain isolated from a human patient. IS 1642 had a size of 1642 bp and contained a single ORF encoding a probable transposase of 503 amino acid residues homologous (79% identity) to that of IS 1549 found in Mycobacterium smegmatis. The IS 1642 included imperfect inverted repeats (5′-cctgacttttatca-3′, 5′-tgataaaagtcggg-3′) on its ends, and was flanked by direct repeats of variable length ranging from 5 to 161 bp. It was suggested that the IS 1642 was widely distributed in many M. avium strains of human patients, and the Southern blot profile of IS 1642 was very diverse among the strains examined. The transposition event of IS 1642 was observed by in vitro repeated passages, showing that the IS 1642 is actually a transposable element. In light of these characteristics, IS 1642 could be a new useful marker when genotyping with high discrimination is required. [ABSTRACT FROM AUTHOR]

SETMAR is a protein lysine methyltransferase that is involved in several DNA processes, including DNA repair via the non-homologous end joining (NHEJ) pathway, regulation of gene expression, illegitimate DNA integration, and DNA decatenation. However, SETMAR is an atypical protein lysine methyltransferase since in anthropoid primates, the SET domain is fused to an inactive DNA transposase. The presence of the DNA transposase domain confers to SETMAR a DNA binding activity towards the remnants of its transposable element, which has resulted in the emergence of a gene regulatory function. Both the SET and the DNA transposase domains are involved in the different cellular roles of SETMAR, indicating the presence of novel and specific functions in anthropoid primates. In addition, SETMAR is dysregulated in different types of cancer, indicating a potential pathological role. While some light has been shed on SETMAR functions, more research and new tools are needed to better understand the cellular activities of SETMAR and to investigate the therapeutic potential of SETMAR. [ABSTRACT FROM AUTHOR]

The eukaryotic Mutator family of transposable elements is widespread in plants. Active or potentially active copies are also found in fungi and protozoans, and sequences related to this family have been detected in metazoans as well. Members of this family are called Mutator-like elements ( MULE s). They encode transposases, which contain a region conserved with transposases of the IS256 prokaryotic family, known to harbor a DDE catalytic domain. Different DDE or D34E motifs have been proposed in some groups of eukaryotic MULEs based on primary sequence conservation. On a large number of protein sequences related to, and representative of, all MULE families, we analyzed global conservation, the close environment of different acidic residues and the secondary structure. This allowed us to identify a potential DDE motif that is likely to be homologous to the one in IS256-like transposases. The characteristics of this motif are depicted in each known family of MULEs. Different hypotheses about the evolution of this triad are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The Tol2 element is a transposon found from a genome of a vertebrate, a small teleost medaka fish. Tol2 encodes a gene for a transposase which is active in vertebrate animals so far tested; for instance, in fish, frog, chicken and mammals, and transgenesis methods using Tol2 have been developed in these model vertebrates. However, it has not been known whether Tol2 can transpose in animals other than vertebrates. Here we report transposition of Tol2 in an invertebrate Drosophila melanogaster. First, we injected a transposon donor plasmid containing a Tol2 construct and mRNA encoding the Tol2 transposase into Drosophila eggs, and found that the Tol2 construct could be excised from the plasmid. Second, we crossed the injected flies, raised the offspring, and found that the Tol2 construct was integrated into the genome of germ cells and transmitted to the next generation. Finally, we constructed a Tol2 construct containing the white gene and injected the transposon donor plasmid and the transposase mRNA into fertilized eggs from the white mutant. We analyzed their offspring, and found that G1 flies with wild type red eyes could be obtained from 35% of the injected fly. We cloned and sequenced 34 integration loci from these lines and showed that these insertions were indeed created through transposition and distributed throughout the genome. Our present study demonstrates that the medaka fish Tol2 transposable element does not require vertebrate-specific host factors for its transposition, and also provides a possibility that Tol2 may be used as a new genetic tool for transgenesis and genome analysis in Drosophila. [Copyright &y& Elsevier]

Horizontal transfer (HT) is central to the evolution of prokaryotic species. Selfish and mobile genetic elements, such as phages, plasmids, and transposons, are the primary vehicles for HTamong prokaryotes. In multicellular eukaryotes, the prevalence and evolutionary significance of HI remain unclear. Here, we identified a set of DNA transposon families dubbed SPACE INVADERS (or SPIN) whose consensus sequences are 96% identical over their entire length (2.9 kb) in the genomes of murine rodents (rat/mouse), bushbaby (prosimian primate), little brown bat (laurasiatherian), tenrec (afrotherian), opossum (marsupial), and two non-mammalian tetrapods (anole lizard and African clawed frog). In contrast, SPIN elements were undetectable in other species represented in the sequence databases, including 19 other mammals with draft whole-genome assemblies. This patchy distribution, coupled with the extreme level of SPIN identity in widely divergent tetrapods and the overall lack of selective constraint acting on these elements, is incompatible with vertical inheritance, but strongly indicative of multiple horizontal introductions. We show that these germline infiltrations likely occurred around the same evolutionary time (15-46 mya) and spawned some of the largest bursts of DNA transposon activity ever recorded in any species lineage (nearly 100,000 SPIN copies per haploid genome in tenrec). The process also led to the emergence of a new gene in the murine lineage derived from a SPIN transposase. In summary, HI of DNA transposons has contributed significantly to shaping and diversifying the genomes of multiple mammalian and tetrapod species. [ABSTRACT FROM AUTHOR]

Aims: To clone and characterize the genes bisdA and bisdB, encoding Ferredoxinbisd (Fdbisd) and cytochrome P450bisd (P450bisd), respectively, from the bisphenol A (BPA) degrading Sphingomonas bisphenolicum strain AO1. Methods and Results: The 3·7 kb region containing bisdA and bisdB was cloned by genome walking and colony hybridization. The deduced N-terminal amino acid sequences of bisdA and bisdB were consistent with those of Fdbisd and P450bisd proteins characterized in our previous report. Two transposase genes, tnpA1 and tnpA2, were also located upstream and downstream of bisdAB. From amino acid sequence analysis, P450bisd has two conserved regions corresponding to the oxygen and heme binding regions of the bacterial cytochrome P450 family. Fdbisd was similar to putidaredoxin-type [2Fe-2S] ferredoxins. Escherichia coli BL21 (DE3) cells bearing bisdB- and bisdAB-recombinant pET19b were able to degrade BPA. A spontaneous mutant, strain AO1L, which was unable to degrade BPA, was isolated from the stock culture, and it was confirmed that strain AO1L had no bisdAB region. Conclusions: P450bisd monooxygenase sytem, encoded by bisdAB, is one system required for BPA hydroxylation in S. bisphenolicum strain AO1. Significance and Impact of the Study: Our results indicate that bisdAB are key genes for BPA degradation in S. bisphenolicum strain AO1. [ABSTRACT FROM AUTHOR]

Conventionally, filamentous fungi are transformed by using conidia or protoplasts as recipients. However, induction of sporulation is difficult in some fungi, and protoplasting is an awing, frequently frustrating, and batch-dependent work. In this study, we established a simple and convenient method to prepare single cells from mycelia without enzymatic protoplasting. As a case study on the pathogenic fungus Phoma herbarum YS4108, the single cells could be directly and highly efficiently transformed with the aid of Tn 5 transposase. The optimal electric pulse delivery parameters were 25 μF in capacitance, 0.75 kV (0.2-cm cuvette) in voltage, and 400 Ω in resistance, under which the efficiency of transposase-assisted transformation (TNAT) was enhanced to two to threefold compared to that of non-TNAT method, resulting in >230 transformants/cuvette (106 recipients). Further cell wall weakening of the single cells by lytic enzymes and linearization of the plasmid were found to have no effects on transformation efficiency, but vector linearization apparently lowered the background growth. The present study for the first time explained that Tn 5 transposase could be used to increase transformation efficiency in filamentous fungi, and the method presented here may be of wide applicability in different studies and may be the first choice when transformation efficiency and convenience are priorities and mycelia have to be used as transformation recipients. [ABSTRACT FROM AUTHOR]

Abstract: The thermostable direct hemolysin coded by the tdh gene is a marker of virulent strains of Vibrio parahaemolyticus. The tdh genes are flanked by insertion sequences collectively named as ISVs or their remnants; but the ISVs so far examined have accumulated mutations in the transposase genes and underwent structural arrangements and their transposition activity could not be expected; the tdh gene was thus considered to have been acquired by V. parahaemolyticus through horizontal transfer in the past during evolution. We recently isolated from the same patient tdh + strains and a tdh − strain (PCR examination) that were otherwise indistinguishable. The purpose of this study was to examine the hypothesis that the tdh − strain was derived from the tdh + strain by a deletion of the tdh gene mediated by a functional ISV. Southern blot hybridization showed tdh + sequences in the tdh − strain (PSU-1466). Nucleotide sequence analysis of the tdh and its flanking sequences revealed the tdh gene was split into two parts and they were located 3182-bp apart in PSU-1466. The two tdh sequences were flanked by one of the ISVs, named as ISVpa3, in PSU-1466. This genetic structure could be explained by an ISVpa3-mediated partial tdh deletion from a tdh + strain followed by transposition of the duplicated ISVpa3 and the deleted tdh sequence into a neighboring location. The ISVpa3 of PSU-1466 coded for a full-length transposase and a DDE motif. We were able to demonstrate transposition activity of the ISVpa3 cloned from PSU-1466 using the replicon fusion assay with the conjugal transfer of a cointegrate from Escherichia coli to V. parahaemolyticus. Our data support ISVpa3-mediated partial tdh deletion resulted in the emergence of the tdh − strain. [Copyright &y& Elsevier]

Abstract: We recently identified a Transposase domain protein called Metnase, which assists in repairing DNA double-strand breaks (DSB) via non-homologous end-joining (NHEJ), and is important for foreign DNA integration into a host cell genome. Since integration is essential for productive lentiviral infection we examined whether Metnase expression levels could have an influence on lentiviral genomic integration. Using cells stably transduced to either over- or under-express Metnase we determined that the expression level of Metnase did indeed correlate with live lentiviral integration. Changes in Metnase levels were accompanied by changes in the number of copies of integrated lentiviral cDNA. While Metnase levels affected lentiviral integration, it had no effect on the amount of either total cellular viral RNA, cDNA or 2-LTR circles. Therefore, Metnase enhances the integration of lentivirus DNA into the host cell genome. [Copyright &y& Elsevier]

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Abstract: A recent study examining genetic variations among the 3 completely sequenced white spot syndrome virus (WSSV) genomes isolated from China, Thailand and Taiwan revealed five major differences among them. Of these differences, a deletion region between ORF 23/24 and a variable region of ORF14/15 prone to recombination were of particular evolutionary significance. Focusing on these regions, 81 WSSV isolates from India were characterized by sequencing polymerase chain reaction (PCR) amplicons. The Indian strains carried a 10,970 bp deletion in the ORF 23/24 region relative to WSSV-TW and WSSV-TH-96-II. Analysis of the ORF 14/15 regions revealed two novel strains of WSSV with unique sequences which could have evolved by recombination. None of the WSSV isolates had a transposase sequence or VP35 gene as reported for Taiwan isolates. The Indian strains were closely related to Thailand strains suggesting movement of a putative ancestor from Thailand to other parts of the world including India. [Copyright &y& Elsevier]

Abstract: Animal genomes contain ∼20,000 genes. Additionally millions of genes for antigen receptors are generated in cells of the immune system from the sets of separate gene segments by a mechanism known as the V(D)J somatic recombination. The components of the V(D)J recombination system, Recombination-Activating Gene proteins (RAG1 and RAG2) and recombination signal sequence (RSS), are thought to have “entered” the vertebrate genome as a hypothetical “RAG transposon”. Recently discovered mobile elements have terminal inverted repeats (TIRs) similar to RSS and may encode proteins with a different degree of similarity to RAG1. We describe a novel N-RAG-TP transposon identified from the sea slug Aplysia californica that encodes a protein similar to the N-terminal part of RAG1 in vertebrates. This refines the “RAG transposon” hypothesis and allows us to propose a scenario for V(D)J recombination machinery evolution from a relic transposon related to the existing mobile elements N-RAG-TP, Chapaev, and Transib. [Copyright &y& Elsevier]

DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3′OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases. [ABSTRACT FROM AUTHOR]

Ancient, inactive copies of transposable elements of the PIF/Harbinger superfamily have been described in vertebrates. We reconstructed components of the Harbinger3̱DR transposon in zebrafish, including a transposase and a second, transposon-encoded protein that has a Myb-like trihelix domain. The reconstructed Harbinger transposon shows efficient cut-and-paste transposition in human cells and preferentially inserts into a 15-bp consensus target sequence. The Myb-like protein is required for transposition and physically interacts with the N-terminal region of the transposase via its C-terminal domain. The Myb-like protein enables transposition in part by promoting nuclear import of the transposase, by directly binding to subterminal regions of the transposon, and by recruiting the transposase to the transposon ends. We investigated the functions of two transposon-derived human proteins: HARBI1, a domesticated transposase-derived protein, and NAIF1, which contains a trihelix motif similar to that described in the Myb-like protein. Physical interaction, subcellular localization, and DNA-binding activities of HARBI1 and NAIF1 suggest strong functional homologies between the Harbinger3̱DR system and their related, host-encoded counterparts. The Harbinger transposon will serve as a useful experimental system for transposon biology and for investigating the enzymatic functions of domesticated, transposon-derived cellular genes. [ABSTRACT FROM AUTHOR]

Galileo is the only transposable element (TE) known to have generated natural chromosomal inversions in the genus Drosophila. It was discovered in Drosophila buzzatii and classified as a Foldback-like element because of its long, internally repetitive, terminal inverted repeats (TIRs) and lack of coding capacity. Here, we characterized a seemingly complete copy of Galileo from the D. buzzatii genome. It is 5,406 bp long, possesses 1,229-bp TIRs, and encodes a 912-aa transposase similar to those of the Drosophila melanogaster 1360 (Hoppel) and P elements. We also searched the recently available genome sequences of 12 Drosophila species for elements similar to Dbuz\Galileo by using bioinformatic tools. Galileo was found in six species (ananassae, willistoni, peudoobscura, persimilis, virilis, and mojavensis) from the two main lineages within the Drosophila genus. Our observations place Galileo within the P superfamily of cut-and-paste transposons and extend considerably its phylogenetic distribution. The interspecific distribution of Galileo indicates an ancient presence in the genus, but the phylogenetic tree built with the transposase amino acid sequences contrasts significantly with that of the species, indicating lineage sorting and/or horizontal transfer events. Our results also suggest that Foldback-like elements such as Galileo may evolve from DNA-based transposon ancestors by loss of the transposase gene and disproportionate elongation of TIRs. [ABSTRACT FROM AUTHOR]

Abstract: PCR analysis of the pandemic strain of Vibrio parahaemolyticus, KX-V237 (total genome sequenced) showed a subculture where the size of the amplicons had increased. The purpose of this study was to analyze the mechanism of this change. We found a 1,243-bp DNA sequence inserted in one of the pandemic marker genes in this strain. The inserted DNA sequence possessed the genetic structures shared by insertion sequences (ISs) of the IS3 family. This IS had 26-bp imperfect terminal inverted repeats (IRs) and two partially overlapping reading frames, orfA and orfB. OrfA codes for a helix-turn-helix, OrfA and OrfAB produced by translational frameshifting code for leucine zipper motifs, and OrfB codes for a DDE motif. orfA and orfB were homologous to those in the IS3 family. This IS was named ISVpa2. Southern blot analysis showed the copy number of ISVpa2 in our stock culture and its subculture of KX-V237 was three and four, respectively; whereas it was only one in the reported genome sequence. Analysis of the flanking sequences for seven ISVpa2 copies showed ISVpa2 is capable of inserting at multiple sites and ISVpa2 causes genetic rearrangements including insertional inactivation of the target gene and adjacent deletion. ISVpa2 created 3-base duplications upon insertion. PCR, hybridization, and nucleotide sequence analyses showed ISVpa2 homologs were detected in all of the 62 other strains of V. parahaemolyticus examined; and in some strains of Vibrio vulnificus (98% identity), Vibrio penaeicida (86% identity), and Vibrio splendidus (87% identity); but was not in 25 other species in the genus Vibrio. The data demonstrate that ISVpa2 is a transpositionally active IS discovered for the first time in V. parahaemolyticus and suggest that ISVpa2 may be transferred among the species of the genus Vibrio. [Copyright &y& Elsevier]

Abstract: Insertion sequences are transposable elements that can represent substantial proportions of prokaryotic genomes and play a substantial role in shaping host genome evolution. As such, evaluating and understanding insertion sequence diversity is an important task to fulfill, because it is expected to yield new insight into the evolution of bacterial transposable elements and contribute to improve genome annotations. Here, I characterized an insertion sequence, termed ISWpi1, for which the taxonomic distribution appears to be restricted to the obligate intracellular alpha-Proteobacterium Wolbachia pipientis. ISWpi1 exhibits ∼46% identity at the amino acid level with members of the IS1031 group of insertion sequences from the IS5 family. However, the IS1031 group is characterized by a transposase gene encoded by a single open reading frame, whereas the ISWpi1 transposase gene consists of two overlapping open reading frames presumably translated as a single protein via programmed translational frameshifting. Such structure suggests that ISWpi1 may instead be related to the IS427 group of insertion sequences from the IS5 family. Altogether, these data indicate that ISWpi1 extends the known spectrum of diversity of the IS5 family, and I propose to define a novel group of insertion sequences within the IS5 family typified by ISWpi1. Probable transpositional activity, relevant insertion site preferences and taxonomic specificity make ISWpi1 a promising tool for experimentally manipulating W. pipientis bacteria, especially in light of the increasing interest in developing these bacteria as tools for controlling insect disease vectors and agricultural pests. [Copyright &y& Elsevier]

Transposable elements are mobile genetic units that exhibit broad diversity in their structure and transposition mechanisms. Transposable elements occupy a large fraction of many eukaryotic genomes and their movement and accumulation represent a major force shaping the genes and genomes of almost all organisms. This review focuses on DNA-mediated or class 2 transposons and emphasizes how this class of elements is distinguished from other types of mobile elements in terms of their structure, amplification dynamics, and genomic effect. We provide an up-to-date outlook on the diversity and taxonomic distribution of all major types of DNA transposons in eukaryotes, including Helitrons and Mavericks. We discuss some of the evolutionary forces that influence their maintenance and diversification in various genomic environments. Finally, we highlight how the distinctive biological features of DNA transposons have contributed to shape genome architecture and led to the emergence of genetic innovations in different eukaryotic lineages. [ABSTRACT FROM AUTHOR]

The tdh (thermostable direct hemolysin) gene occurs in some strains of Vibrio species. All tdh genes are flanked by insertion sequence-like elements (ISV). All previous attempts have failed to detect transposition of these ISVs. In this work, we have built a transposition detection system in E. coli and succeeded in detecting the transposition of an insertion sequence-like element at a frequency of Kmr mutants of 7.2 × 10−6. A specific flanking sequence (5′-Py-Pu-3′) was found on either side of the target duplication. [ABSTRACT FROM AUTHOR]

A new family, termed TxpB, of DNA transposons belonging to the piggyBac superfamily was found in 3 Xenopus species (Xenopus tropicalis, Xenopus laevis, and Xenopus borealis). Two TxpB subfamilies of Kobuta and Uribo1 were found in all the 3 species, and another subfamily termed Uribo2 was found in X. tropicalis. Molecular phylogenetic analyses of their open reading frames (ORFs) revealed that TxpB transposons have been maintained for over 100 Myr. Both the Uribo1 and the Uribo2 ORFs were present as multiple copies in each genome, and some of them were framed by terminal inverted repeat sequences. In contrast, all the Kobuta ORFs were present as a single copy in each genome and exhibited high evolutionary conservation, suggesting domestication of Kobuta genes by the host. Genomic insertion polymorphisms of the Uribo1 and Uribo2 transposons (nonautonomous type) were observed in a single species of X. tropicalis, indicating recent transposition events. Transfection experiments in cell culture revealed that an expression vector construct for the intact Uribo2 ORF caused precise excision of a nonautonomous Uribo2 element from the target vector construct but that for the Kobuta ORF did not. The present results support our viewpoint that some Uribo2 members are naturally active autonomous transposons, whereas Kobuta members may be domesticated by hosts. [ABSTRACT FROM PUBLISHER]

Abstract: The higher-order DNA–protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end–enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1–E interaction is essential to promote transpososome assembly, R3–E interaction contributes to the native topology of the transpososome, and L1–E and R2–E interactions are not required for assembly. The data on L2–E and L3–E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R–E and perhaps one L–E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions. [Copyright &y& Elsevier]

Abstract: Mariner-like elements (MLEs) are class-II transposable elements that move within the genome of their hosts by means of a DNA-mediated “cut and paste” mechanism. MLEs have been identified in several organisms, from most of the phyla. Nevertheless, only a few of the sequences characterized contain an intact open reading frame. Investigation of the genome of a coastal crab, Pachygrapsus marmoratus, has identified nine Pacmmar elements, two of which have an open reading frame encoding a putatively functional transposase. Nucleic acid analyses and comparison with the previous data showed that the GC contents of MLEs derived from coastal organisms such as P. marmoratus are significantly higher than those of terrestrial MLEs and significantly lower than those of hydrothermal ones. Furthermore, molecular phylogeny analyses have shown that Pacmmar elements constitute a new lineage of the irritans subfamily within the mariner family. [Copyright &y& Elsevier]

Virus-based gene therapy has advanced to clinical trials; however, this approach may result in serious adverse events including oncogenesis and the possibility of triggering fatal immune responses. Nonviral gene delivery approaches have a better safety profile, but their in vivo application has been largely limited in the past due to their inefficient delivery into cells and lack of stable chromosomal integration that is necessary for long-term therapeutic benefit. However, recent advances suggest that the use of Sleeping Beauty transposons, a novel integrating nonviral vector system, are capable of achieving long-lasting therapeutic levels of transgene expression in preclinical settings. These observations and other ongoing relevant studies may unlock the therapeutic potential of nonviral gene therapy for human diseases. iubmb Life, 59: 1 - 6, 2007 [ABSTRACT FROM AUTHOR]

Abstract: Site-specific recombinases are important tools for genomic engineering in many living systems. Applications of recombinases are, however, constrained by the DNA targeting endemic of the recombinase used. A tremendous range of recombinase applications can be envisioned if the targeting of recombinase specificity can be made readily programmable. To address this problem we sought to generate zinc finger-recombinase fusion proteins (RecZFs) capable of site-specific function in a diversity of genetic contexts. Our first RecZF, Tn3Ch15X2, recombined substrates derived from the native Tn3 resolvase recombination site. Substrate Linked Protein Evolution (SLiPE) was used to optimize the catalytic domains of the enzymes Hin, Gin, and Tn3 for resolution between non-homologous sites. One of the evolved clones, GinL7C7, catalyzed efficient, site-specific recombination in a variety of sequence contexts. When introduced into human cells by retroviral transduction, GinL7C7 excised a 1.4 kb EGFP cassette out of the genome, diminishing fluorescence in ∼17% of transduced cells. Following this template of rational design and directed evolution, RecZFs may eventually mediate gene therapies, facilitate the genetic manipulation of model organisms and cells, and mature into powerful new tools for molecular biology and medicine. [Copyright &y& Elsevier]

Abstract: The Tn5 transposase is an example of a class of proteins that move DNA sequences (transposons) via a process called transposition. DNA transposition is a widespread genetic mobility mechanism that has profoundly affected the genomes of nearly all organisms. We have used single-DNA micromanipulation experiments to study the process by which Tn5 DNA transposons are identified and processed by their transposase protein. We have determined that the energy barrier to disassemble catalytically active synaptic complexes is 16 kcal mol−1. However, we have found that the looping organization of DNA segments by transposase is less sequence-driven than previously thought. Loops anchored at some non-transposon end sequences display a disassembly energy barrier of 14 kcal mol−1, nearly as stable as the synapses formed at known transposon end sequences. However, these non-transposon end sequence independent complexes do not mediate DNA cleavage. Therefore, the sequence-sensitivity for DNA binding and looping by Tn5 transposase is significantly less than that required for DNA cleavage. These results have implications for the in vivo down regulation of transposition and the cis-transposition bias of transposase. [Copyright &y& Elsevier]

The rearrangement of antigen receptor genes is initiated by double- strand breaks catalyzed by the RAG1/2 complex at the junctions of recombination signal sequences and coding segments. As with some "cut-and-paste" transposases, such as Tn5 and Hermes, a DNA hairpin is formed at one end of the break via a nicked intermediate. By using abasic DNA substrates, we show that different base positions are important for the two steps of cleavage. Removal of one base in the coding flank enhances hairpin formation, bypassing a requirement for a paired complex of two signal sequences. Rescue by abasic substrates is consistent with a base-flip mechanism seen in the crystal structure of the Tn5 postcleavage complex and may mimic the DNA changes on paired complex formation. We have searched for a tryptophan residue in RAG1 that would be the functional equivalent of W298 in Tn5, which stabilizes the DNA interaction by stacking the flipped base on the indole ring. A W956A mutation in RAG1 had an inhibitory effect on both nicking and hairpin stages that could be rescued by abasic substrates. W956 is therefore a likely candidate for interacting with this base during hairpin formation. [ABSTRACT FROM AUTHOR]

Abstract: DNA transposition reactions typically involve a strand transfer step wherein the transposon ends are covalently joined by the transposase protein to a short target site. There is very little known about the transposase-DNA interactions that direct this process, and thus our overall understanding of the dynamics of DNA transposition reactions is limited. Tn5 presents an attractive system for defining such interactions because it has been possible to solve the structure of at least one Tn5 transposition intermediate: a transpososome formed with pre-cleaved ends. However, insertion specificity in the Tn5 system is low and this has hampered progress in generating target-containing transpososomes that are homogeneous in structure (i.e. where a single target site is engaged) and therefore suitable for biochemical and structural analysis. We have developed a system where the Tn5 transpososome integrates almost exclusively into a single target site within a short DNA fragment. The key to establishing this high degree of insertion specificity was to use a target DNA with tandem repeats of a previously characterized Tn5 insertion hotspot. The target DNA requirements to form this strand transfer complex are evaluated. In addition, we show that target DNAs missing single phosphate groups at specific positions are better substrates for strand transfer complex formation relative to the corresponding unmodified DNA fragments. Moreover, utilization of missing phosphate substrates can increase the degree of target site selection. A method for concentrating and partially purifying the Tn5 strand transfer complex is described. [Copyright &y& Elsevier]

Tn10 is a bacterial transposon that transposes through a non-replicative mechanism. This mode of DNA transposition is widely used in bacteria and is also used by “DNA-based” transposons in eukaryotes. Tn10 has served as a paradigm for this mode of transposition and continues to provide novel insights into how steps in transposition reactions occur and how these steps are regulated. A common feature of transposition reactions is that they require the formation of a higher order protein-DNA complex called a transpososome. A major objective in the last few years has been to better understand the dynamics of transpososome assembly and progression through the course of transposition reactions. This problem is particularly interesting in the Tn10 system because two important host proteins, IHF and H-NS, have been implicated in regulating transpososome assembly and/or function. Interestingly, H-NS is an integral part of stress response pathways in bacteria, and its function is known to be sensitive to changes in environmental conditions. Consequently, H-NS may provide a means of allowing Tn10 to responed to changing environmental conditions. The current review focuses on the roles of both IHF and H-NS on Tn10 transposition. [ABSTRACT FROM AUTHOR]

Abstract: A DNA methyl-binding column was used to isolate genomic fragments enriched for DNA-methylation from Aspergillus parasiticus. One of the isolated sequences presented 67% identity at the protein level with the transposase from the transposable element Tan1 of Aspergillus niger var. awamori, and was found to be present in at least 20 copies in the Aspergillus oryzae database. Analysis of four copies showed evidence of C:G to T:A transitions in at least 98.2% of the mutations found over a 1032–1180bp region spanning a large part of the transposase sequence. Using copy specific primers three sequences were amplified from a different strain of A. oryzae and a similar pattern of C:G to T:A transitions was found. These transitions are similar to those observed in RIP, in Neurospora crassa, where cytosine-methylation is believed to be involved. Using methylation-sensitive Southern blotting, no evidence of methylation was found in the transposase sequences in these two A. oryzae strains as well as one A. parasiticus and one Aspergillus flavus strain. [Copyright &y& Elsevier]

Belonging to Class II of transposable elements, En/Spm transposons are widespread in a variety of distantly related plant species. Here, we report on the sequence conservation of the transposase region from sequence analyses of En/Spm-like transposons from Poaceae species, namely Zingeria biebersteiniana, Zingeria trichopoda, Triticum monococcum, Triticum urartu, Hordeum spontaneum, and Aegilops speltoides. The transposase region of En/Spm-like transposons was cloned, sequenced, and compared with equivalent regions of Oryza and Arabidopsis from the gene bank database. Southern blot analysis indicated that the En/Spm transposon was present in low ( Hordeum spontaneum, Triticum monococcum, Triticum urartu) through medium ( Zingeria bieberstiana, Zingeria trichopoda) to relatively high ( Aegilops speltoides) copy numbers in Poaceae species. A cytogenetic analysis of the chromosomal distribution of En/Spm transposons revealed the concurence of the chromosomal localization of the En/Spm clusters with mobile clusters of rDNA. An analysis of En/Spm-like transposase amino acid sequences was carried out to investigate sequence divergence between 5 genera — Triticum, Aegilops, Zingeria, Oryza and Arabidopsis. A distance matrix was generated; apparently, En/Spm-like transposase sequences shared the highest sequence homology intra-generically and, as expected, these sequences were significantly diverged from those of O. sativa and A. thaliana. A sequence comparison of En/Spm-like transposase coding regions defined that the intra-genomic complex of En/Spm-like transposons could be viewed as relatively independent, vertically transmitted, and permanently active systems inside higher plant genomes. The sequence data from this article was deposited in the EMBL/GenBank Data Libraries under the accession nos. AY707995-AY707996-AY707997-AY707998-AY707999-AY708000-AY708001-AY708002-AY708003-AY708004-AY708005-AY708005-AY265312. [ABSTRACT FROM AUTHOR]

A transposon-based random mutation library of AcMNPV, the type species of baculovirus, was constructed using a Tn5 transposon. The green fluorescence protein gene under the control of the Drosophila hsp 70 promoter was inserted into the transposon for easy tracking in insect cells. In vitro transposition was carried out using the transposon and AcMNPV genomic DNA to allow the random insertion of the transposon into the virus genome. The transposed genome was then used to transfect Sf21 insect cells, and a library of mutant viruses capable of expressing green fluorescence protein was obtained. Two mutant viruses, B9F and Li6A were isolated, and the sites of transposon insertion were determined to be within the coding regions of the 94k and p10 genes, respectively. Both genes were determined to be nonessential in viral replication and infection. This technique will be very useful in the functional study of baculovirus genes. [ABSTRACT FROM AUTHOR]

We present the crystal structure of the catalytic domain of Mos1 transposase, a member of the Tc1/mariner family of transposases. The structure comprises an RNase H-like core, bringing together an aspartic acid triad to form the active site, capped by N- and C-terminal α-helices. We have solved structures with either one Mg2+ or two Mn2+ ions in the active site, consistent with a two-metal mechanism for catalysis. The lack of hairpin-stabilizing structural motifs is consistent with the absence of a hairpin intermediate in Mos1 excision. We have built a model for the DNA-binding domain of Mos1 transposase, based on the structure of the bipartite DNA-binding domain of Tc3 transposase. Combining this with the crystal structure of the catalytic domain provides a model for the paired-end complex formed between a dimer of Mos1 transposase and inverted repeat DNA. The implications for the mechanisms of first and second strand cleavage are discussed. [ABSTRACT FROM AUTHOR]

Summary: Very few CACTA transposon-like sequences have been described in Solanaceae species. Sequence information has been restricted to partial transposase (TPase)-like fragments, and no target gene of CACTA-like transposon insertion has been described in tomato to date. In this manuscript, we report on a CACTA transposon-like insertion in intron I of tomato (Lycopersicon esculentum) invertase gene Lin5 and TPase-like sequences of several Solanaceae species. Consensus primers deduced from the TPase region of the tomato CACTA transposon-like element allowed the amplification of similar sequences from various Solanaceae species of different subfamilies including Solaneae (Solanum tuberosum), Cestreae (Nicotiana tabacum) and Datureae (Datura stramonium). This demonstrates the ubiquitous presence of CACTA-like elements in Solanaceae genomes. The obtained partial sequences are highly conserved, and allow further detection and detailed analysis of CACTA-like transposons throughout Solanaceae species. CACTA-like transposon sequences make possible the evaluation of their use for genome analysis, functional studies of genes and the evolutionary relationships between plant species. [Copyright &y& Elsevier]

Phytoplasmas are cell wall-less prokaryotes living as obligate parasites and pathogens of plants and insects, making them attractive subjects for studies to gain a greater understanding of transkingdom parasitism and pathogenicity. During a study of two phytoplasma genomes, we obtained evidence for previously unreported clustering of genes, pseudogenes, mobile genetic elements, intergenic repeat units, and repetitive extragenic palindromes that occur in multiple, homologous clusters in some phytoplasma genomes. The clusters represent previously unrecognized mosaics, possibly assembled through multiple events of targeted mobile element attack, duplication, recombination, and rearrangement. Multiple clusters could conceivably afford potential for genome reduction through homologous recombination. Differences in the sizes and multiplicity of such clusters possibly account for some of the previously reported but unexplained variations in genome size among closely related phytoplasma strains. [ABSTRACT FROM AUTHOR]

Mariner-like elements ( MLEs) belong to the Tc1-mariner superfamily of DNA transposons, which is very widespread in animal genomes. We report here the first complete description of a MLE, Xtmar1, within the genome of a poikilotherm vertebrate, the amphibian Xenopus tropicalis. A close relative, XlMLE, is also characterized within the genome of a sibling species, Xenopus laevis. The phylogenetic analysis of the relationships between MLE transposases reveals that Xtmar1 is closely related to Hsmar2 and Bytmar1 and that together they form a second distinct lineage of the irritans subfamily. All members of this lineage are also characterized by the 36- to 43-bp size of their imperfectly conserved inverted terminal repeats and by the –8-bp motif located at their outer extremity. Since XlMLE, Xlmar1, and Hsmar2 are present in species located at both extremities of the vertebrate evolutionary tree, we looked for MLE relatives belonging to the same subfamily in the available sequencing projects using the amino acid consensus sequence of the Hsmar2 transposase as an in silico probe. We found that irritans MLEs are present in chordate genomes including most craniates. This therefore suggests that these elements have been present within chordate genomes for 750 Myr and that the main way they have been maintained in these species has been via vertical transmission. The very small number of stochastic losses observed in the data available suggests that their inactivation during evolution has been very slow. [ABSTRACT FROM AUTHOR]

Aims: To compare genetic composition of plasmids using microarrays composed of randomly selected fragments of plasmid DNA. Methods and Results: Separate shotgun libraries were constructed from plasmid DNA pooled from Escherichia coli and Salmonella enterica. Cloned fragments were used as probes for microarrays. Plasmid targets were labelled, hybridized overnight, and bound targets were imaged after enzymatic signal amplification. Control hybridizations demonstrated significantly higher signal when probes and targets shared >95% sequence identity. Diagnostic sensitivity and specificity of the assay was 95 and 99%, respectively. Cluster analysis showed close matches for replicate experiments with a high correlation between replicates ( r = 0·91) compared with the correlation for nonreplicates ( r = 0·09). Analysis of hybridization data from 43 plasmids generated five distinct clusters, two for known serovar-specific plasmids, one for enterohemorrhagic E. coli plasmids, and two for plasmids harboring a recently disseminated antibiotic resistance gene ( blaCMY-2). Conclusion: Mixed-plasmid microarrays are suitable for comparing genetic content of wild-type plasmids and hybridization results from this study suggest several novel hypotheses about plasmid gene exchange between E. coli and S. enterica. Significance and Impact of Study: Mixed-plasmid microarrays permit rapid, low cost analysis and comparison of many plasmids. This ability is critical to understanding the source, fate, and transport of plasmids amongst commensal and pathogenic bacteria. [ABSTRACT FROM AUTHOR]