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The use of transposase in cell line development (CLD) programs has experienced increased popularity over the past decade. However, few studies have described the mechanism of action and the genomic and phenotypic characteristics of clones derived from transposase. Additionally, how these traits impact long-term bioproduction is unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to characterize the unique fingerprints associated with transposase-derived clones. Transposase reduces the cellular pool of transient vector as early as three days post transfection following transfection and expedites stable pool establishment by up to two weeks. Furthermore, recombinant DNA expression is significantly improved up to ∼3 fold along with a greater balance of antibody heavy and light chain transcripts, resulting in higher titers in transposase generated pools. Transposase derived pools contained an often innumerable number of integration sites, representing a vast increase in integration site diversity over randomly generated pools, which were bottlenecked at 1-3 integration sites per pool. These transposase mediated integrations typically occurred in clean singlets, free of genomic scars such as deletions, inversions, and other modifications associated with legacy transfection methods which exhibited higher copy numbers per integration site. Relative declines in gene expression occur with copy number increase in the randomly generated, but not the transposase derived clones. Furthermore, transposase-derived clones were more likely to exhibit enhanced a long term stability profile, including product quality attributes such as mannose-5. This improved stability may result from circumventing mechanisms associated with the silencing of tandem repeats. Thus, transposase-mediated approaches can provide multifaceted molecular and phenotypic advantages in cell line development when compared to legacy random-integration methods., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S. Huhn, B. Jiang, M. Chang, X. Tang, Z. Du are employees of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and potentially own stock and/or hold stock options in Merck & Co., Inc., Kenilworth, NJ, USA. Merck & Co., Inc provided support in the form of salaries for all authors but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the “author contributions” section., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Although new genes can arrive from modes other than duplication, few examples are well characterized. Given high expression in some human brain subregions and a putative link to psychological disorders [e.g., schizophrenia (SCZ)], suggestive of brain functionality, here we characterize piggyBac transposable element-derived 1 (PGBD1). PGBD1 is nonmonotreme mammal-specific and under purifying selection, consistent with functionality. The gene body of human PGBD1 retains much of the original DNA transposon but has additionally captured SCAN and KRAB domains. Despite gene body retention, PGBD1 has lost transposition abilities, thus transposase functionality is absent. PGBD1 no longer recognizes piggyBac transposon-like inverted repeats, nonetheless PGBD1 has DNA binding activity. Genome scale analysis identifies enrichment of binding sites in and around genes involved in neuronal development, with association with both histone activating and repressing marks. We focus on one of the repressed genes, the long noncoding RNA NEAT1, also dysregulated in SCZ, the core structural RNA of paraspeckles. DNA binding assays confirm specific binding of PGBD1 both in the NEAT1 promoter and in the gene body. Depletion of PGBD1 in neuronal progenitor cells (NPCs) results in increased NEAT1/paraspeckles and differentiation. We conclude that PGBD1 has evolved core regulatory functionality for the maintenance of NPCs. As paraspeckles are a mammal-specific structure, the results presented here show a rare example of the evolution of a novel gene coupled to the evolution of a contemporaneous new structure., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Mutants of Micrococcus luteus strain ATCC49732 lacking the yellow pigment sarcinaxanthin were observed at an unexpectedly high frequency and the molecular basis was investigated. PCR probing revealed complete deletion of the crt biosynthetic operon in 11/14 mutants. Inverse PCR was used to identify a common breakpoint 35 kb downstream from crt precisely at the end of the right inverted repeat (IRR) of a partial ISMlu8 element that lies between two inversely oriented full-length ISMlu2. A total of three different breakpoints 5' to crt were found with the sequence CTAG one bp 5' to each novel junction. Analysis of 35 genomic sites with single ISMlu8 insertions showed that ISMlu8 transposase has high specificity for CTAG, implicating its key role in formation of the Δcrt deletions. No downstream deletion endpoints were observed at an immediately adjacent ISMlu8 with a nearly identical IRR in the same orientation and slightly closer to the crt operon, indicating that access of ISMlu8 transposase to the ISMlu2-flanked ISMlu8 IRR is greatly enhanced by the surrounding oppositely oriented ISMlu2s. The association of high frequency genomic rearrangement with this distinctive natural configuration of ISs from two different IS families offers a new insight into IS element evolutionary potential., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

The development of highly productive, genetically stable manufacturing cell lines is on the critical path to IND filing for protein-based biologic drugs. Here, we describe the Leap-In Transposase® platform, a novel transposon-based mammalian (e.g., Chinese hamster ovary) cell line development system that produces high-titer stable pools with productivity and product quality attributes that are highly comparable to clones that are subsequently derived therefrom. The productivity distributions of clones are strongly biased toward high producers, and genetic and expression stability is consistently high. By avoiding the poor integration rates, concatemer formation, detrimental transgene recombination, low average expression level, unpredictable product quality, and inconsistent genetic stability characteristic of nonhomologous recombination methods, Leap-In provides several opportunities to de-risk programs early and reduce timelines and resources., (© 2021 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

TcBuster is a hAT-family DNA transposon from the red flour beetle, Tribolium castaneum. The TcBuster transposase is of interest for genome engineering as it is highly active in insect and mammalian cells. To test the predicted catalytic triad of TcBuster, each residue of the catalytic triad of a haemagglutinin-tagged TcBuster transposase was individually mutated to a structurally conserved amino acid. Using a drug-resistant colony assay for transposon integration, we found that the D223N, D289N, and E589Q mutants of TcBuster transposase were inactive in human cells. We used a modified chromatin immunoprecipitation assay to determine that each mutant maintained binding to TcBuster transposon inverted repeat elements. Although the catalytic mutants retained their transposon binding properties, mutants displayed altered expression and localization in human cells. None of the catalytic mutants formed characteristic TcBuster transposase rodlet structures, and the D223N and D289N mutants were not able to be detected by immunofluorescence microscopy. Immunoblot analysis demonstrated that the E589Q mutant is less abundant than wild-type TcBuster transposase. Cells transfected with either TcBuster or TcBuster-E589Q transposase were imaged by structured illumination microscopy to quantify differences in the length of the transposase rodlets. The average length of the TcBuster transposase rodlets (N = 39) was 3.284 μm while the E589Q rodlets (N = 33) averaged 1.157 μm (p < 0.0001; t-test). The catalytic triad mutations decreased overall protein levels and disrupted transposase rodlet formation while nuclear localization and DNA binding to the inverted repeat elements were maintained. Our results may have broader implications for the overproduction inhibition phenomenon observed for DNA transposons., (Published by Elsevier Inc.)

IS 26 forms cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS 26 and some close relatives, IS 1006 , IS 1008 , and a natural hybrid, IS 1006/IS1008 , which are found predominantly in Acinetobacter spp., to interact was examined. IS 1006/1008 consists of 175 bp from IS 1006 at the left end, with the remainder from IS 1008. These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other's ends. In a recA -negative Escherichia coli strain, IS 1006 , IS 1008 , and IS 1006/1008 each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS 26 However, using mixed pairs, targeted cointegration was detected only when IS 1008 was paired with the IS 1006 / 1008 hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS 26 paired with IS 26 :: catA1 , an artificially constructed IS 26 derivative that includes the catA1 gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed. IMPORTANCE The IS 26 family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS 26 is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS 26 seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS 26 family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS 26 , IS 257/ IS 431 , and IS 1216 , which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS 26 family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS 26 and naturally occurring close relatives of IS 26 that are found near resistance genes in Acinetobacter baumannii and are widespread in Acinetobacter species., (Copyright © 2021 Harmer and Hall.)

Sleeping Beauty (SB) is a transposon system that has been widely used as a genetic engineering tool. Central to the development of any transposon as a research tool is the ability to integrate a foreign piece of DNA into the cellular genome. Driven by the need for efficient transposon-based gene vector systems, extensive studies have largely elucidated the molecular actors and actions taking place during SB transposition. Close transposon relatives and other recombination enzymes, including retroviral integrases, have served as useful models to infer functional information relevant to SB. Recently obtained structural data on the SB transposase enable a direct insight into the workings of this enzyme. These efforts cumulatively allowed the development of novel variants of SB that offer advanced possibilities for genetic engineering due to their hyperactivity, integration deficiency, or targeting capacity. However, many aspects of the process of transposition remain poorly understood and require further investigation. We anticipate that continued investigations into the structure-function relationships of SB transposition will enable the development of new generations of transposition-based vector systems, thereby facilitating the use of SB in preclinical studies and clinical trials.

In comparison with retrotransposons, DNA transposons make up a smaller proportion of most plant genomes. However, these elements are often proximal to genes to affect gene expression depending on the activity of the transposons, which is largely reflected by the activity of the transposase genes. Here, we show that three AT-rich introns were retained in the TNP2-like transposase genes of the Bot1 (Brassica oleracea transposon 1) CACTA transposable elements in Brassica oleracea, but were lost in the majority of the Bot1 elements in Brassica rapa. A recent burst of transposition of Bot1 was observed in B. oleracea, but not in B. rapa. This burst of transposition is likely related to the activity of the TNP2-like transposase genes as the expression values of the transposase genes were higher in B. oleracea than in B. rapa. In addition, distinct populations of small RNAs (21, 22 and 24 nt) were detected from the Bot1 elements in B. oleracea, but the vast majority of the small RNAs from the Bot1 elements in B. rapa are 24 nt in length. We hypothesize that the different activity of the TNP2-like transposase genes is likely associated with the three introns, and intron loss is likely reverse transcriptase mediated. Furthermore, we propose that the Bot1 family is currently undergoing silencing in B. oleracea, but has already been silenced in B. rapa. Taken together, our data provide new insights into the differentiation of transposons and their role in the asymmetric evolution of these two closely related Brassica species., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

A mutant excision + /integration - piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.

Bamboo, a fast-growing non-timber forest plant with many uses, is a valuable species for green development. However, bamboo flowering is very infrequent, extending, in general, for up to 120 years. Ecologically, bamboo species are generally better adapted to various environments than other grasses. Therefore, the species deserves a special status in what could be called Ecological Bioeconomy. An understanding of the genetic processes of bamboo can help us sustainably develop and manage bamboo forests. Transposable elements (TEs), jumping genes or transposons, are major genetic elements in plant genomes. The rapid development of the bamboo reference genome, at the chromosome level, reveals that TEs occupy over 63.24% of the genome. This is higher than found in rice, Brachypodium, and sorghum. The bamboo genome contains diverse families of TEs, which play a significant role in bamboo's biological processes including growth and development. TEs provide important clues for understanding the evolution of the bamboo genome. In this chapter, we briefly describe the current status of research on TEs in the bamboo genome, their regulation, and transposition mechanisms. Perspectives for future research are also provided.

The majority of eukaryotic genomes contain a large fraction of repetitive sequences that primarily originate from transpositional bursts of transposable elements (TEs). Repbase serves as a database for eukaryotic repetitive sequences and has now become the largest collection of eukaryotic TEs. During the development of Repbase, many new superfamilies/lineages of TEs, which include Helitron, Polinton, Ginger and SINEU, were reported. The unique composition of protein domains and DNA motifs in TEs sometimes indicates novel mechanisms of transposition, replication, anti-suppression or proliferation. In this review, our current understanding regarding the diversity of eukaryotic TEs in sequence, protein domain composition and structural hallmarks is introduced and summarized, based on the classification system implemented in Repbase. Autonomous eukaryotic TEs can be divided into two groups: Class I TEs, also called retrotransposons, and Class II TEs, or DNA transposons. Long terminal repeat (LTR) retrotransposons, including endogenous retroviruses, non-LTR retrotransposons, tyrosine recombinase retrotransposons and Penelope-like elements, are well accepted groups of autonomous retrotransposons. They share reverse transcriptase for replication but are distinct in the catalytic components responsible for integration into the host genome. Similarly, at least three transposition machineries have been reported in eukaryotic DNA transposons: DDD/E transposase, tyrosine recombinase and HUH endonuclease combined with helicase. Among these, TEs with DDD/E transposase are dominant and are classified into 21 superfamilies in Repbase. Non-autonomous TEs are either simple derivatives generated by internal deletion, or are composed of several units that originated independently.

Mariner-like elements (MLE) are a super-family of DNA transposons widespread in animal and plant genomes. Based on their transposition characteristics, such as random insertions and high-frequency heterogeneous transpositions, several MLEs have been developed to be used as tools in gene tagging and gene therapy. Two active MLEs, Ppmar1 and Ppmar2 , have previously been identified in moso bamboo ( Phyllostachys edulis ). Both of these have a preferential insertion affinity to AT-rich region and their insertion sites are close to random in the host genome. In Ppmar2 element, we studied the affinities of terminal inverted repeats (TIRs) to DNA binding domain (DBD) and their influence on the transposition activity. We could identify two putative boxes in the TIRs which play a significant role in defining the TIR's affinities to the DBD. Seven mutated TIRs were constructed, differing in affinities based on similarities with those of other plant MLEs. Gel mobility shift assays showed that the TIR mutants with mutation sites G669A-C671A had significantly higher affinities than the mutants with mutation sites C657T-A660T. The high-affinity TIRs indicated that their transposition frequency was 1.5-2.0 times higher than that of the wild type TIRs in yeast transposition assays. The MLE mutants with low-affinity TIRs had relatively lower transposition frequency from that of wild types. We conclude that TIR affinity to DBD significantly affects the transposition activity of Ppmar2 . The mutant MLEs highly active TIRs constructed in this study can be used as a tool for bamboo genetic studies.

Transposable elements (TEs) are ubiquitous in both prokaryotes and eukaryotes, and the dynamic character of their interaction with host genomes brings about numerous evolutionary innovations and shapes genome structure and function in a multitude of ways. In traditional classification systems, TEs are often being depicted in simplistic ways, based primarily on the key enzymes required for transposition, such as transposases/recombinases and reverse transcriptases. Recent progress in whole-genome sequencing and long-read assembly, combined with expansion of the familiar range of model organisms, resulted in identification of unprecedentedly long transposable units spanning dozens or even hundreds of kilobases, initially in prokaryotic and more recently in eukaryotic systems. Here, we focus on such oversized eukaryotic TEs, including retrotransposons and DNA transposons, outline their complex and often combinatorial nature and closely intertwined relationship with viruses, and discuss their potential for participating in transfer of long stretches of DNA in eukaryotes., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Transposable elements (TE), small mobile genetic elements unable to exist independently of the host genome, were initially believed to be exclusively deleterious genomic parasites. However, it is now clear that they play an important role as bacterial mutagenic agents, enabling the host to adapt to new environmental challenges and to colonize new niches. This review focuses on the impact of insertion sequences (IS), arguably the smallest TE, on bacterial genome plasticity and concomitant adaptability of phenotypic traits, including resistance to antibacterial agents, virulence, pathogenicity and catabolism. The direct consequence of IS transposition is the insertion of one DNA sequence into another. This event can result in gene inactivation as well as in modulation of neighbouring gene expression. The latter is usually mediated by de-repression or by the introduction of a complete or partial promoter located within the element. Furthermore, transcription and transposition of IS are affected by host factors and in some cases by environmental signals offering the host an adaptive strategy and promoting genetic variability to withstand the environmental challenges.

We demonstrated the possibility of obtaining insertion mutants by a modified technique using EZ::TN5 system during culturing of the recipient strain on a dense nutrient medium and exclusion of the centrifugation stage. The frequency of transposon mutants of E. coli 10979/EZ::TN5 was 2×10 -6 . Genetically modified strains were characterized by kanamycin resistance, inability to L-malate assimilation, changes in the expression of individual proteins of protein mass-spectra (5096.3, 6252.9, and 9067.7 Da), and the presence of fragments in genomic DNA amplified by specific forward and reverse primers that were homologous to Tn5 transposon insertion sites. The modified procedure for obtaining insertion mutants by using EZ::TN5 system was not inferior by the efficiency to the standard procedure, but shortens experiment duration, simplifies it, and reduces the risks related to working with group 2 pathogenicity microorganisms.

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Insertion sequences are the simplest intracellular Mobile Genetic Elements which can occur in very high numbers in prokaryotic genomes, where they play an important evolutionary role by promoting genome plasticity. As such, the studies on the diversity and distribution of insertion sequences in genomes not yet investigated can contribute to improve the knowledge on a bacterial species and to identify new transposable elements. The present work describes the occurrence of insertion sequences in Lactococcus garvieae, an opportunistic emerging zoonotic and human pathogen, also associated with different food matrices. To date, no insertion elements have been described for L. garvieae in the IS element database. The analysis of the twelve published L. garvieae genomes identified 15 distinct insertion sequences that are members of the IS3, IS982, IS6, IS21 and IS256 families, including five new elements. Most of the insertion sequences in L. garvieae show substantial homology to the Lactococcus lactis elements, suggesting the movement of IS between these two species phylogenetically closely related. ISLL6 elements belonging to IS3 family were most abundant, with several copies distributed in 9 of the 12 genomes analyzed. An alignment analysis of two complete genomes carrying multi-copies of this insertion sequence indicates a possible involvement of ISLL6 in chromosomal rearrangement., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Insertion sequence (IS) elements are short, self-replicating DNA sequences that are capable of efficiently spreading over the host genome. Possessing varied integration specificity IS elements are capable of the irreversible inactivation of genes, which diversifies the pool of intact genetic determinants in host populations. In the current study, we performed a complex analysis of IS elements (Vsa IS) in the previously sequenced genome of Aliivibrio salmonicida LFI1238 and proposed a model of the spread of the Vsa IS elements over the genome of this microorganism. Along with the prediction of the integration sites for Vsa IS elements, the current study provides an overview of the properties of A. salmonicida IS elements, as well as information regarding their occurrence in different bacterial classes. An analysis of individual alleles of the IS elements has allowed us to depict a history of the accumulation of mutations and to describe distinctive microevolution lines for actively transposing Vsa IS elements in the genome of A. salmonicida LFI1238. Our results demonstrate the high importance of the dead end microevolution of actively transposing Vsa IS elements for the inactivation of genes in A. salmonicida LFI1238., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Tolerance of the foodborne pathogen Listeria monocytogenes to sublethal concentrations of disinfectants has been frequently reported. Particularly, quaternary ammonium compounds (QACs) such as benzalkonium chloride (BC) are often used in disinfectants and also as antiseptics in food industry and hospitals. Recently, we described Tn6188, a novel transposon in L. monocytogenes harbouring the transporter QacH, a molecular mechanism leading to increased tolerance to BC. In this study, we investigated the presence of Tn6188 within the genus Listeria spp. Our screening indicates that the distribution of Tn6188 may be limited to L. monocytogenes. We confirm that QacH is responsible for the observed increase in tolerance by complementation of a qacH deletion mutant and introducing qacH in a Tn6188 negative strain. We investigated the transporter's substrate spectrum by determining minimal inhibitory concentrations (MICs) and showed that QacH also confers higher tolerance towards other QACs and ethidium bromide (EtBr). This result was supported by increased expression of qacH in the presence of the various substrates as determined by quantitative reverse transcriptase PCR (qRT-PCR). In addition, we detected expression of a Tn6188 transposase gene and circular forms of Tn6188, suggesting activity and possible transfer of this transposon., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen., (© 2014 by The Mycological Society of America.)

Insertion sequences (ISs) are small transposable elements widespread in bacterial genomes, where they play an essential role in chromosome evolution by stimulating recombination and genetic flow. Despite their ubiquity, it is unclear how ISs interact with the host. Here, we report a survey of the orientation patterns of ISs in bacterial chromosomes with the objective of gaining insight into the interplay between ISs and host chromosomal functions. We find that a significant fraction of IS families present a consistent and family-specific orientation bias with respect to chromosomal DNA replication, especially in Firmicutes. Additionally, we find that the transposases of up to nine different IS families with different transposition pathways interact with the β sliding clamp, an essential replication factor, suggesting that this is a widespread mechanism of interaction with the host. Although we find evidence that the interaction with the β sliding clamp is common to all bacterial phyla, it also could explain the observed strong orientation bias found in Firmicutes, because in this group β is asymmetrically distributed during synthesis of the leading or lagging strands. Besides the interaction with the β sliding clamp, other asymmetries also play a role in the biased orientation of some IS families. The utilization of the highly conserved replication sliding clamps suggests a mechanism for host regulation of IS proliferation and also a universal platform for IS dispersal and transmission within bacterial populations and among phylogenetically distant species.

The goldfish (Carassius auratus) Tgf2 transposon is a vertebrate DNA transposon that belongs to the hAT transposon family. In this study, we constructed plasmids containing either the full-length Tgf2 transposon (pTgf2 plasmid) or a partially-deleted Tgf2 transposon (ΔpTgf2 plasmid), and microinjected these plasmids into fertilized zebrafish (Danio rerio) eggs at the one- to two-cell stage. DNA extracted from the embryos was analyzed by PCR to assess transient excision, if any, of the exogenous plasmid and to verify whether Tgf2 is an autonomous transposon. The results showed that excision-specific bands were not detected in embryos injected with the ΔpTgf2 plasmid, while bands of 300-500bp were detected in embryos injected with pTgf2, which indicated that the full-length Tgf2-containing plasmid could undergo autonomous excision in zebrafish embryos. DNA cloned from 24 embryos injected with pTgf2 was sequenced, and the results suggested that Tgf2 underwent self-excision in zebrafish embryos. Cloning and PCR analysis of DNA extracted from embryos co-injected with ΔpTgf2 and in vitro-transcribed transposase mRNA indicated that partially-deleted-Tgf2-containing ΔpTgf2 plasmid also underwent excision, in the presence of functional transposase mRNA. DNA cloned from 25 embryos co-injected with ΔpTgf2 and transposase mRNA was sequenced, and the results suggested that partially-deleted Tgf2 transposons plasmids were excised. These results demonstrated that excisions of Tgf2 transposons were mediated by the Tgf2 transposase, which in turn confirmed that Tgf2 is an autonomous transposon., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Transposable elements (TEs) are major components of both prokaryotic and eukaryotic genomes and play a significant role in their evolution. In this study, we have identified new prokaryotic DDE transposase families related to the eukaryotic Mutator-like transposases. These genes were retrieved by cascade PSI-Blast using as initial query the transposase of the streptococcal integrative and conjugative element (ICE) TnGBS2. By combining secondary structure predictions and protein sequence alignments, we predicted the DDE catalytic triad and the DNA-binding domain recognizing the terminal inverted repeats. Furthermore, we systematically characterized the organization and the insertion specificity of the TEs relying on these prokaryotic Mutator-like transposases (p-MULT) for their mobility. Strikingly, two distant TE families target their integration upstream σA dependent promoters. This allowed us to identify a transposase sequence signature associated with this unique insertion specificity and to show that the dissymmetry between the two inverted repeats is responsible for the orientation of the insertion. Surprisingly, while DDE transposases are generally associated with small and simple transposons such as insertion sequences (ISs), p-MULT encoding TEs show an unprecedented diversity with several families of IS, transposons, and ICEs ranging in size from 1.1 to 52 kb.

The hAT superfamily comprises a large and diverse array of DNA transposons found in all supergroups of eukaryotes. Here we characterized the Drosophila buzzatii BuT2 element and found that it harbors a five-exon gene encoding a 643-aa putatively functional transposase. A phylogeny built with 85 hAT transposases yielded, in addition to the two major groups already described, Ac and Buster, a third one comprising 20 sequences that includes BuT2, Tip100, hAT-4&#95;BM, and RP-hAT1. This third group is here named Tip. In addition, we studied the phylogenetic distribution and evolution of BuT2 by in silico searches and molecular approaches. Our data revealed BuT2 was, most often, vertically transmitted during the evolution of genus Drosophila being lost independently in several species. Nevertheless, we propose the occurrence of three horizontal transfer events to explain its distribution and conservation among species. Another aspect of BuT2 evolution and life cycle is the presence of short related sequences, which contain similar 5' and 3' regions, including the terminal inverted repeats. These sequences that can be considered as miniature inverted repeat transposable elements probably originated by internal deletion of complete copies and show evidences of recent mobilization.

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this. In this work we test the hypothesis that FB-NOF is an active transposon nowadays. We search for its expression product, obtaining its cDNA, and propose the ORF and the sequence of its potential protein. We found that the NOF protein is not a transposase as it lacks any of the motifs of known transposases and also shows structural homology with hydrolases, therefore FB-NOF cannot belong to the superfamily MuDR/foldback, as up to now it has been classified, and can be considered as a non-autonomous transposable element. The alignment with the published genomes of 12 Drosophila species shows that NOF presence is restricted only to the 6 Drosophila species belonging to the melanogaster group., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The transposon BuT5 caused two chromosomal inversions fixed in two Drosophila species of the repleta group, D. mojavensis and D. uniseta. BuT5 copies are approximately 1-kb long, lack any coding capacity, and do not resemble any other transposable element (TE). Because of its elusive features, BuT5 has remained unclassified to date. To fully characterize BuT5, we carried out bioinformatic similarity searches in available sequenced genomes, including 21 Drosophila species. Significant hits were only recovered for D. mojavensis genome, where 48 copies were retrieved, 22 of them approximately 1-kb long. Polymerase chain reaction (PCR) and dot blot analyses on 54 Drosophila species showed that BuT5 is homogeneous in size and has a widespread distribution within the repleta group. Thus, BuT5 can be considered as a miniature inverted-repeat TE. A detailed analysis of the BuT5 hits in D. mojavensis revealed three partial copies of a transposon with ends very similar to BuT5 and a P-element-like transposase-encoding region in between. A putatively autonomous copy of this P element was isolated by PCR from D. buzzatii. This copy is 3,386-bp long and possesses a seven-exon gene coding for an 822-aa transposase. Exon-intron boundaries were confirmed by reverse transcriptase-PCR experiments. A phylogenetic tree built with insect P superfamily transposases showed that the D. buzzatii P element belongs to an early diverging lineage within the P-element family. This divergent P element is likely the master transposon mobilizing BuT5. The BuT5/P element partnership probably dates back approximately 16 Ma and is the ultimate responsible for the generation of the two chromosomal inversions in the Drosophila repleta species group.