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Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here, we analyzed the transposable elements (TEs) and found that they comprise approximately 76% of the V. speciosa genome, thus being the most abundant type of DNA sequence in this gigantic genome. The V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons, which constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated con-tigs against the V. speciosa TE database allowed for the identification of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, the TE composition in V. speciosa resembles the TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated with past climate crises. Key words: climate crisis, demographic changes, endangered species, ferns, genome size, relict populations, tetraploidy, transposable elements, Vandenboschia speciosa. Le Vandenboschia speciosa est une espece menacee de fougere tetraploide a grand genome (10,5 Gb). Sa repartition geographique se caracterise par des refuges floraux tertiaires disjoints, avec des populations reliques qui ont survecu a des crises climatiques anterieures. Dans ce travail, les auteurs analysent les elements transpos-ables (TE) et rapportent qu'ils constituent environ 76 % du genome du V. spieciosa, ils representent ainsi le type de sequence d'ADN le plus abondant dans ce genome gigantesque. Le genome du V. spieciosa est compose a 51 % et a 5,6 % respectivement d'elements de Classe I et de Classe II. Les retrotransposons a LTR etaient les TE les plus abond-ants chez cette espece (au moins 42 % de genome), suivis des retrotransposons non-LTR, lesquels constituaient aumoins 8,7 % du genome de cette espece. Les auteurs ont realise une analyse additionnelle pour identifier la nature des elements non-annotes (19 % du genome). Une analyse BLAST des contigs non-annotes contre une base de don-nees des TE du V. spieciosa a permis d'en identifier pres de la moitie, lesquels constituent vraisemblablement des variants divergents par rapport aux TE annotes. De maniere generale, la composition en TE du V. speciosa ressem-ble a la composition rencontree chez les plantes a graines. De plus, cette caracterisation des sequences repetees suggere que trois episodes d'amplification de tous les TE se seraient produits, vraisemblablement en raison de changements demographiques associes aux crises climatiques passees. Mots-cles: crise climatique, changements demographiques, espece menacees, fougeres, taille du genome, populations reliques, tetraplo'idie, elements transposables, Vandenboschia speciosa., Introduction Transposable elements (TEs) are ubiquitous components of eukaryotic genomes that are considered drivers of genome evolution (Bohne et al. 2008; Belyayev 2014; Bourque et al. 2018), and have a [...]

Author(s): Nian Liu [1]; Cameron H. Lee [2]; Tomek Swigut [1]; Edward Grow [2]; Bo Gu [1]; Michael C. Bassik (corresponding author) [2, 3]; Joanna Wysocka (corresponding author) [1, 4, [...]

Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1-RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire of antibodies and T cell receptors, was a pivotal event in the evolution of the adaptive immune system of jawed vertebrates. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of the amphioxus ProtoRAG transposase (an evolutionary relative of RAG), we identify amino acid residues and domains the acquisition or loss of which underpins the propensity of RAG for coupled cleavage, its preference for asymmetric DNA substrates and its inability to perform transposition in cells. In particular, we identify two adaptations specific to jawed-vertebrates--arginine 848 in RAG1 and an acidic region in RAG2--that together suppress RAG-mediated transposition more than 1,000-fold. Our findings reveal a two-tiered mechanism for the suppression of RAG-mediated transposition, illuminate the evolution of V(D)J recombination and provide insight into the principles that govern the molecular domestication of transposons. Identification of the changes that converted a transposase to a recombinase sheds light on the evolution of the vertebrate adaptive immune system., Author(s): Yuhang Zhang [sup.1] , Tat Cheung Cheng [sup.2] , Guangrui Huang [sup.3] , Qingyi Lu [sup.3] , Marius D. Surleac [sup.4] , Jeffrey D. Mandell [sup.1] , Pierre Pontarotti [...]

The R gene of soybean, presumably encoding a MYB transcription factor, controls seed coat color. The gene consists of multiple alleles, R (black), r-m (black spots and (or) concentric streaks on brown seed), and r (brown seed). This study was conducted to determine the structure of the MYB transcription factor gene in a near-isogenic line (NIL) having r-m allele. PCR amplification of a fragment of the candidate gene Glyma.09G235100 generated a fragment of about 1 kb in the soybean cultivar Clark, whereas a fragment of about 14 kb in addition to fragments of 1 and 1.4 kb were produced in L72-2040, a Clark 63 NIL with the r-m allele. Clark 63 is a NIL of Clark with the rxp and Rps1 alleles. A DNA fragment of 13 060 bp was inserted in the intron of Glyma.09G235100 in L72-2040. The fragment had the CACTA motif at both ends, imperfect terminal inverted repeats (TIR), inverse repetition of short sequence motifs close to the 5' and 3' ends, and a duplication of three nucleotides at the site of integration, indicating that it belongs to a CACTA-superfamily transposable element. We designated the element as Tgm11. Overall nucleotide sequence, motifs of TIR, and subterminal repeats were similar to those of Tgml and Tgsl, suggesting that these elements comprise a family. Keywords: CACTA-superfamily transposable element, Glycine max, R gene, seed coat color, soybean. Le gene R chez le soya, lequel code vraisemblablement pour un facteur de transcription MYB, determine la couleur de la graine. Le gene compte plusieurs alleles, dont R (noir), r-m (taches noires ou stries concentriques sur un fond brun) et r (brun). Ce travail a ete realise pour determiner la structure de ce gene chez une lignee quasi-isogenique (NIL) possedant un allele r-m. L'amplification PCR d'un fragment du gene candidat, Glyma.09G235100 a produit un fragment d'environ 1 kb chez le cultivar Clark, tandis qu'un fragment d'environ 14 kb, en plus de fragments de 1 et de 1,4 kb, ont ete obtenus chez L72-2040, une NIL derivee de Clark 63 ayant l'allele r-m. Clark 63 est elle-meme une NIL de Clark et possedant les alleles rxp et Rps1. Un fragment d'ADN de 13 060 pb etait insere dans l'intron du Glyma.09G235100 chez L72-2040. Ce segment presentait un motif CACTA aux deux extremites, des inversions terminales inversees (TIR) imparfaites, une repetition inversee de courts motifs situes pres des extremites 5' et 3', ainsi qu'une duplication de trois nucleotides a l'endroit du site d'integration; toutes des caracteristiques indiquant qu'il s'agit d'un element transposable de la superfamille CACTA. Les auteurs ont nomme cet element Tgm11. Globalement, la sequence nucleotidique, les motifs des TIR et les repetitions subterminales etaient semblables a celles rencontrees chez Tgm1 et Tgs1, ce qui suggere que ces elements forment une famille. [Traduit par la Redaction] Mots-cles : element transposable de la superfamille CACTA, Glycine max, gene R, couleur de la graine, soya., Introduction Transposable elements are DNA fragments that can change their positions in the genome, occasionally resulting in disruption of genes, or variegation of tissues when inserted in genes responsible for [...]

The generation of widespread epigenetically activated short interfering RNAs by the targeting of microRNAs to transposon transcripts in Arabidopsis thaliana is shown to be a latent mechanism that only becomes active when the transcripts are epigenetically reactivated, for example, during reprogramming of the germ line. Transposon targeting by plant miRNAs In the model plant Arabidopsis, transposable elements are generally silenced by DNA methylation. Here, Robert Martienssen and colleagues describe an alternative silencing mechanism. Targeting occurs in the absence of DNA methylation, and generates 21-nucleotide short interfering RNAs -- which the authors call 'easiRNA', for epigenetically activated siRNA -- of a type that has been observed previously in pollen grains. Thousands of transposons are targeted by at least 50 microRNAs, most of them conserved in plant evolution. The authors propose that microRNA-directed easiRNA biogenesis specifically targets transposon transcripts when they are epigenetically reactivated during germline reprogramming. In plants, post-transcriptional gene silencing (PTGS) is mediated by DICER-LIKE 1 (DCL1)-dependent microRNAs (miRNAs), which also trigger 21-nucleotide secondary short interfering RNAs (siRNAs) via RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), DCL4 and ARGONAUTE 1 (AGO1).sup.1,2,3, whereas transcriptional gene silencing (TGS) of transposons is mediated by 24-nucleotide heterochromatic (het)siRNAs, RDR2, DCL3 and AGO4 (ref. 4). Transposons can also give rise to abundant 21-nucleotide 'epigenetically activated' small interfering RNAs (easiRNAs) in DECREASED DNA METHYLATION 1 (ddm1) and DNA METHYLTRANSFERASE 1 (met1) mutants, as well as in the vegetative nucleus of pollen grains.sup.5 and in dedifferentiated plant cell cultures.sup.6. Here we show that easiRNAs in Arabidopsis thaliana resemble secondary siRNAs, in that thousands of transposon transcripts are specifically targeted by more than 50 miRNAs for cleavage and processing by RDR6. Loss of RDR6, DCL4 or DCL1 in a ddm1 background results in loss of 21-nucleotide easiRNAs and severe infertility, but 24-nucleotide hetsiRNAs are partially restored, supporting an antagonistic relationship between PTGS and TGS. Thus miRNA-directed easiRNA biogenesis is a latent mechanism that specifically targets transposon transcripts, but only when they are epigenetically reactivated during reprogramming of the germ line. This ancient recognition mechanism may have been retained both by transposons to evade long-term heterochromatic silencing and by their hosts for genome defence., Author(s): Kate M. Creasey [sup.1] , Jixian Zhai [sup.2] , Filipe Borges [sup.1] , Frederic Van Ex [sup.1] , Michael Regulski [sup.1] , Blake C. Meyers [sup.2] , Robert A. [...]

Insertions of mobile elements.sup.1-4, mitochondrial DNA.sup.5 and fragments of nuclear chromosomes.sup.6 at DNA double-strand breaks (DSBs) threaten genome integrity and are common in cancer.sup.7-9. Insertions of chromosome fragments at V(D)J recombination loci can stimulate antibody diversification.sup.10. The origin of insertions of chromosomal fragments and the mechanisms that prevent such insertions remain unknown. Here we reveal a yeast mutant, lacking evolutionarily conserved Dna2 nuclease, that shows frequent insertions of sequences between approximately 0.1 and 1.5 kb in length into DSBs, with many insertions involving multiple joined DNA fragments. Sequencing of around 500 DNA inserts reveals that they originate from Ty retrotransposons (8%), ribosomal DNA (rDNA) (15%) and from throughout the genome, with preference for fragile regions such as origins of replication, R-loops, centromeres, telomeres or replication fork barriers. Inserted fragments are not lost from their original loci and therefore represent duplications. These duplications depend on nonhomologous end-joining (NHEJ) and Pol4. We propose a model in which alternative processing of DNA structures arising in Dna2-deficient cells can result in the release of DNA fragments and their capture at DSBs. Similar DNA insertions at DSBs are expected to occur in any cells with linear extrachromosomal DNA fragments.A deficiency of Dna2 causes duplication of DNA fragments and their insertion into the genome at double-stranded breaks in yeast., Author(s): Yang Yu [sup.1] , Nhung Pham [sup.1] , Bo Xia [sup.2] [sup.3] [sup.4] , Alma Papusha [sup.1] , Guangyu Wang [sup.2] [sup.3] [sup.4] , Zhenxin Yan [sup.1] , Guang [...]

Transposable elements (TEs) are among the most abundant components of many eukaryotic genomes. Efforts to explain TE abundance, as well as TE diversity among genomes, have led some researchers to draw an analogy between genomic and ecological processes. Adopting this perspective, we conducted an analysis of the cow (Bos taurus) genome using techniques developed by community ecologists to determine whether environmental factors influence community composition. Specifically, each chromosome within the Bos taurus genome was treated as a 'linear transect', and a multivariate redundancy analysis (RDA) was used to identify large-scale spatial patterns in TE communities associated with 10 TE families. The position of each TE community on the chromosome accounted for ~50% of the variation along the chromosome 'transect'. Multivariate analysis further revealed an effect of gene density on TE communities that is influenced by several other factors in the (genomic) environment, including chromosome length and TE density. The results of this analysis demonstrate that ecological methods can be applied successfully to help answer genomic questions. Key words: genome ecology, transposon ecology, transposable elements, spatial patterns, gene density, chromosome length. Les elements transposables (TE) sont parmi les composantes les plus abondantes au sein de plusieurs genomes eucaryotes. Les efforts visant a expliquer cette abondance et diversite des TE au sein des genomes ont amene certains chercheurs a voir une analogie entre les processus genomiques et evolutifs. C'est dans cette perspective que les auteurs ont realise une analyse du genome de la vache (Bos taurus) en employant des techniques developpees par les ecologistes des communautes pour determiner si des facteurs environnementaux influencent la composition d'une communaute. Specifiquement, chaque chromosome au sein du genome du Bos taurus a ete traite comme un <> et une analyse de redondance (RDA) multivariee a ete realisee pour identifier les grandes lignes de la repartition de communautes de TE appartenant a 10 familles de TE. La position de chaque communaute de TE au sein du chromosome expliquait ~50 % de la variation le long d'un'transect' chromosomique. Une analyse multivariee a ensuite revele un effet de la densite genique sur les communautes de TE qui est influence par plusieurs autres composantes de l'environnement (genomique), dont la longueur du chromosome et la densite en TE. Les resultats de cette analyse montrent que les methodes de l'ecologie peuvent servir a repondre a des questions genomiques. [Traduit par la Redaction] Mots-cles : ecologie du genome, ecologie des transposons, elements transposables, distribution spatiale, densite genique, longueur du chromosome., Introduction The biology of transposable elements Transposable elements (TEs) are mobile segments of DNA capable of being cut or copied from one location in the genome and reinserted into another. [...]

The research field of comparative genomics is moving from a focus on genes to a more holistic view including the repetitive complement. This study aimed to characterize relative proportions of the repetitive fraction of large, complex genomes in a nonmodel system. The monocotyledonous plant order Asparagales (onion, asparagus, agave) comprises some of the largest angiosperm genomes and represents variation in both genome size and structure (karyotype). Anonymous, low coverage, single-end Illumina data from 11 exemplar Asparagales taxa were assembled using a de novo method. Resulting contigs were annotated using a reference library of available monocot repetitive sequences. Mapping reads to contigs provided rough estimates of relative proportions of each type of transposon in the nuclear genome. The results were parsed into general repeat types and synthesized with genome size estimates and a phylogenetic context to describe the pattern of transposable element evolution among these lineages. The major finding is that although some lineages in Asparagales exhibit conservation in repeat proportions, there is generally wide variation in types and frequency of repeats. This approach is an appropriate first step in characterizing repeats in evolutionary lineages with a paucity of genomic resources. Key words: Asparagales, transposable elements, genome size evolution, genome assembly, comparative genomics, monocots. La genomique comparee est un domaine de recherche dont le point focal quitte les genes pour adopter une vue plus holistique du genome incluant le complement de sequences repetees. Ce travail visait a caracteriser les proportions relatives des sequences repetees au sein de grands genomes complexes chez un systeme non-modele. Chez les plantes, au sein des monocotyledones, l'ordre des Asparagales (oignon, asperge, agave) compte certains des plus grands genomes d'angiospermes et presente de la variation tant pour la taille du genome que pour sa structure (caryotype). Des sequences Illumina anonymes offrant une faible couverture chez 11 taxons representatifs des Asparagales ont ete assemblees au moyen d'une approche de novo. Les contigs obtenus ont ete annotes a l'aide d'une banque de sequences repetees presentes chez les monocotyledones. L'alignement des sequences sur ces contigs a permis de determiner approximativement les proportions relatives de chaque type de transposon au sein du genome nucleaire. Ces sequences ont ete separees en plusieurs grands groupes de sequences repetees et analysees a la lumiere de la taille des genomes et de leur contexte phylogenetique pour decrire revolution des elements transposables au sein de ces embranchements. L'observation la plus importante est que, bien que la proportion du genome constituee de sequences repetees soit conservee chez certains embranchements des Asparagales, il existe une variation considerable quant aux types et frequences de ces elements repetes. L'approche adoptee dans ce travail constitue une premiere etape appropriee pour caracteriser les sequences repetees au sein d'embranchements dotes de peu de ressources genomiques. [Traduit par la Redaction] Mots-cles: Asparagales, elements transposables, evolution de la taille du genome, assemblage de genomes, genomique comparee, monocotyledones., Introduction The advent of next generation sequencing (NGS) technology is allowing unprecedented access to genomic information, including not just genes but also the repetitive fraction of genomes (Gregory 2005). Increasing [...]

Eukaryotic organisms have dynamic genomes, with transposable elements (TEs) as a major contributing factor. Although the large autonomous TEs can significantly shape genomic structures during evolution, genomes often harbor more miniature nonautonomous TEs that can infest genomic niches where large TEs are rare. In spite of their cut-and-paste transposition mechanisms that do not inherently favor copy number increase, miniature inverted-repeat transposable elements (MITEs) are abundant in eukaryotic genomes and exist in high copy numbers. Based on the large number of MITE families revealed in previous studies, accurate annotation of MITEs, particularly in newly sequenced genomes, will identify more genomes highly rich in these elements. Novel families identified from these analyses, together with the currently known families, will further deepen our understanding of the origins, transposase sources, and dramatic amplification of these elements. Key words: transposable elements, miniature inverted-repeat transposable elements, genome, evolution, copy number. Les organismes eucaryotes possedent des genomes dynamiques dont les elements transposables (TE) forment une composante importante. Bien que les TE autonomes de grande taille puissent fa^onner la structure d'un genome au cours de revolution, les genomes contiennent souvent davantage de TE miniatures non-autonomes qui peuvent occuper des niches genomiques ou les grands TE sont rares. En depit de leur mecanisme de transposition de type couper-coller qui ne favorise pas intrinsequement un accroissement du nombre de copies, les elements transposables miniatures a inversions repetees (MITE) sont abondants au sein des genomes eucaryotes et sont presents en un grand nombre de copies. En se fondant sur le grand nombre de familles de MITE deja identifiees, une annotation soignee des MITE, particulierement chez les genomes nouvellement sequences, permettra d'identifier d'autres genomes tres riches en ces elements. De nouvelles familles identifiees au cours de ces analyses, ajoutees aux familles deja connues, permettront d'ameliorer nos connaissances sur les origines, les sources de transposases et l'amplification dramatique de ces elements. [Traduit par la Redaction] Mots-cles : elements transposables, elements transposables miniatures a inversions repetees, genome, evolution, nombre de copies., Introduction Transposable elements (TEs) are widely distributed among both prokaryotic and eukaryotic genomes. They are capable of moving from one chromosome location to another, a process called transposition, and thereby [...]

The retrotransposons HeT-A, TART, and TAHRE, which maintain Drosophila telomeres, transpose specifically onto chromosome ends to form long arrays that extend the chromosome and compensate for terminal loss. Because they transpose by target-primed reverse transcription, each element is oriented so that its 5' end serves as the extreme end of the chromosome until another element transposes to occupy the terminal position. Thus 5' sequences are at risk for terminal erosion while the element is at the chromosome end. Here we report that TART elements in Drosophila melanogaster and Drosophila virilis show species-specific innovations in promoter architecture that buffer loss of sequence exposed at chromosome ends. The two elements have evolved different ways to effect this protection. The D. virilis TART ([TART.sup.vir]) promoter is found in the 3' UTR of the element directly upstream of the element transcribed. Transcription starts within the upstream element so that a 'Tag' of extra sequence is added to the 5' end of the newly transcribed RNA. This Tag provides expendable sequence to buffer end erosion of essential 5' sequence after the RNA is reverse transcribed onto the chromosome. In contrast, the D. melanogaster TART ([TART.sup.mel]) promoter initiates transcription deep within the 5' UTR, but the element is able to replace and extend the 5' UTR sequence by copying sequence from its 3' UTR, we believe while being reverse transcribed onto the chromosome end. Astonishingly, end-protection in [TART.sup.vir] and [HeT-A.sup.mel] are essentially identical (using Tags), whereas [HeT-A.sup.vir] is clearly protected from end erosion by an as-yet-unspecified program. chromosome end protection | promoter evolution | pseudo-LTR promoter | perfect nonterminal repeat | antisense RNA doi/ 10.1073/pnas.1015926107

Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 [lambda]pir and S17-1 [lambda]pir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 [lambda]pir and S17-1 [lambda]pir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of II-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria. doi: 10.1128/JB.00621-10

Pseudomonas aeruginosa is a major human opportunistic pathogen, especially for patients in intensive care units or with cystic fibrosis. Multidrug resistance is a common feature of this species. In a previous study we detected the ant(4')-IIb gene in six multiresistant clinical isolates of P. aeruginosa, and determination of the environment of the gene led to characterization of Tn6061. This 26 586 bp element, a member of the Tn3 family of transposons, carried 10 genes conferring resistance to six drug classes. The ant(4')-IIb sequence was flanked by directly repeated copies of ISCR6 in all but one of the strains studied, consistent with ISCR6-mediated gene acquisition. Tn6061 was chromosomally located in six strains and plasmid-borne in the remaining isolate, suggesting horizontal acquisition. Duplication-insertion of IS6100, that ended Tn6061, was responsible for large chromosomal inversions. Acquisition of Tn6061 and chromosomal inversions are further examples of intricate mechanisms that contribute to the genome plasticity of P. aeruginosa. DOI 10.1099/mic.0.033639-0

Transposon mutagenesis has been applied to a hyper-invasive clinical isolate of Campylobacter jejuni, 01/51. A random transposon mutant library was screened in an in vitro assay of invasion and 26 mutants with a significant reduction in invasion were identified. Given that the invasion potential of C. jejuni is relatively poor compared to other enteric pathogens, the use of a hyper-invasive strain was advantageous as it greatly facilitated the identification of mutants with reduced invasion. The location of the transposon insertion in 23 of these mutants has been determined; all but three of the insertions are in genes also present in the genome-sequenced strain NCTC 11168. Eight of the mutants contain transposon insertions in one region of the genome (~14 kb), which when compared with the genome of NCTC 11168 overlaps with one of the previously reported plasticity regions and is likely to be involved in genomic variation between strains. Further characterization of one of the mutants within this region has identified a gene that might be involved in adhesion to host cells. DOI 10.1099/mic.0.033399-0

The non-LTR retrotransposons forming Drosophila telomeres constitute a robust mechanism for telomere maintenance, one which has persisted since before separation of the extant Drosophila species. These elements in D. melanogaster differ from nontelomeric retrotransposons in ways that give insight into general telomere biology. Here, we analyze telomere-specific retrotransposons from D. virilis, separated from D. melanogaster by 40 to 60 million years, to evaluate the evolutionary divergence of their telomeric traits. The telomeric retrotransposon HeT-A from D. melanogaster has an unusual promoter near its 3' terminus that drives not the element in which it resides, but the adjacent downstream element in a head-to-tail array. An obvious benefit of this promoter is that it adds nonessential sequence to the 5' end of each transcript, which is reverse transcribed and added to the chromosome. Because the 5' end of each newly transposed element forms the end of the chromosome until another element transposes onto it, this nonessential sequence can buffer erosion of sequence essential for HeT-A. Surprisingly, we have now found that HeT-A in D. virilis has a promoter typical of non-LTR retrotransposons. This promoter adds no buffering sequence; nevertheless, the complete 5' end of the element persists in telomere arrays, necessitating a more precise processing of the extreme end of the telomere in D. virilis. telomeres | retrotransposons | end replication problem | promoter evolution doi/ 10.1073/pnas.1000612107

Abscisic acid (ABA) is one of the most important phytohormones involved in abiotic stress responses, seed maturation, germination, and senescence. ABA is predominantly produced in vascular tissues and exerts hormonal responses in various cells, including guard cells. Although ABA responses require extrusion of ABA from ABA-producing cells in an intercellular ABA signaling pathway, the transport mechanisms of ABA through the plasma membrane remain unknown. Here we isolated an ATP-binding cassette (ABC) transporter gene, AtABCG25, from Arabidopsis by genetically screening for ABA sensitivity. AtABCG25 was expressed mainly in vascular tissues. The fluorescent protein-fused AtABCG25 was localized at the plasma membrane in plant cells. In membrane vesicles derived from AtABCG25-expressing insect cells, AtABCG25 exhibited ATP-dependent ABA transport. The AtABCG25-overexpressing plants showed higher leaf temperatures, implying an influence on stomatal regulation. These results strongly suggest that AtABCG25 is an exporter of ABA and is involved in the intercellular ABA signaling pathway. The presence of the ABA transport mechanism sheds light on the active control of multicellular ABA responses to environmental stresses among plant cells. Arabidopsis | ABCG | transposon-tagged lines doi/10.1073/pnas.0912516107

The bacterial transposon Tn917 inserts preferentially in the terminus region of some members of the Firmicutes. To determine what molecular process was being targeted by the element, we analyzed Tn917 target site selection in Bacillus subtilis. We find that Tn917 insertions accumulate around the central terminators, terI and terII, in wild-type cells with or without the SP[beta] lysogen. Highly focused targeting around terI and terlI requires the trans-acting termination protein RTP, but it is unaffected in strains compromised in dimer resolution or chromosome translocation. This work indicates that Tn917 is sensitive to differences in DNA replication termination between the Firmicutes. doi: 10.1128/JB.01023-09

Mycoplasma genitalium is the smallest self-replicating organism and a successful human pathogen associated with a range of genitourinary maladies. As a consequence of its restricted genome size, genes that are highly conserved in other bacteria are absent in M. genitalium. Significantly, genes that encode antioxidants like superoxide dismutase and catalase-peroxidase are lacking. Nevertheless, comparative genomics has revealed that MG 454 of M. genitalium encodes a protein with putative function as an organic hydroperoxide reductase (Ohr). In this study, we found that an M. genitalium transposon mutant that lacks expression of MG_454 was sensitive to killing by t-butyl hydroperoxide and cumene hydroperoxide. To understand whether this sensitivity to hydroperoxides was linked to MG_454, we cloned this gene behind an arabinose-inducible PBAD promoter in plasmid pHERD20T and transformed this construct (pHERDMG454) into a Pseudomonas aeruginosa strain having deletion in its ohr gene (ohr mutant) and showing sensitivity to organic hydroperoxides. The P. aeruginosa ohr mutant harboring pHERDMG454, when induced with arabinose, was able to reverse its sensitivity to organic hydroperoxides, thus supporting the notion that the product of MG_454 resists organic hydroperoxides in M. genitalium. Surprisingly, real-time reverse transcription-PCR showed that expression of MG_454 in M. genitalium was not elevated in response to oxidative stress but was elevated in response to physical stresses, like salt (NaCl) and heat. Although failure of MG_454 to respond to oxidative stress in M. genitalium implies the absence of a known oxidative stress response regulator in the genome of M. genitalium, elevated expression of MG_454 due to physical stress suggests its control by an unidentified regulator. doi: 10.1128/JB.01066-08

Many human colonic Bacteroides spp. harbor a conjugative transposon, CTnDOT, which carries two antibiotic resistance genes, tetQ and ermF. A distinctive feature of CTnDOT is that its excision and transfer are stimulated by tetracycline. Regulation of the genes responsible for excision has been described previously. We provide here the first characterization of the regulation of CTnDOT transfer (tra) genes. Reverse transcription-PCR analysis of the region containing the tra genes showed that these genes are regulated at the transcriptional level. Surprisingly, increased production of tra gene mRNA in tetracycline-stimulated cells was mediated by the proteins encoded by the excision genes. Previous studies have shown that expression of the excision gene operon is controlled by the regulatory protein RteC. Accordingly, it was possible that RteC was also regulating tra gene expression and that the excision proteins were only accessory proteins. However, placing the excision gene operon under the control of a heterologous promoter showed that the excision proteins alone could activate tra gene expression and that RteC was not directly involved. We also found a second level of tra gene control. The transfer of CTnDOT was inhibited by a DNA segment that included only a portion of the 3' end of one of the excision genes (exc). This segment contained a small open reading frame, rteR. By replacing the codons encoding the first two amino acids of the putative protein product of this open reading frame with stop codons, we showed that the rteR gene might encode a small regulatory RNA. RteR acted in trans to reduce the number of tra transcripts in a way that was independent of the excision proteins. The repressive effect of RteR was not the result of decreased stability of the tra mRNA. Instead, RteR appears to be modulating the level of tra gene expression in some more direct fashion. The complex regulatory system that controls and links the expression of CTnDOT excision and transfer genes may be designed to ensure stable maintenance of CTnDOT in nature by reducing the fitness toll it takes on the cell that harbors it. doi: 10.1128/JB.00739-09

Here, we show that recombinant Drosophila elp1 (D-elp1) produced in Sf9 cells or Escherichia coil, corresponding to the largest of the three subunits in the RNA polymerase II core elongator complex, has RNA-dependent RNA polymerase (RdRP) activity. D-elp1 is a noncanonical RdRP that can synthesize dsRNA from different ssRNA templates using either a primer-dependent or primer-independent initiation mechanism. Of the three core subunits, only D-elp1 depletion inhibits RNAi in S2 cells but does not affect micro RNA function. Furthermore, D-elp1 depletion results in increased steady state levels of representative transposon RNAs and a decrease in the corresponding transposon antisense transcripts and endo siRNAs. In contrast, although Dcr-2 depletion results in increased transposon RNA levels and a reduction in the corresponding endo siRNAs, there is no change in the transposon antisense RNA levels. In D-elp1 null third instar larvae transposon RNA levels are also increased and the corresponding transposon antisense RNAs are reduced. D-elp1 associates tightly with Dcr-2, similar to the Dicer-RdRP interaction observed in lower eukaryotes. These results identify an aspect of the RNAi pathway in Drosophila that suggest transposon derived endo siRNAs, critical for transposon suppression, are produced, in part, in a D-elp1 dependent step that converts transposon RNA into dsRNA that is subsequently processed by Dcr-2. The generality of this mechanism in genome defense and RNA silencing in higher eukaryotes is suggested. elongator | genome defence | cellular stress

Mycobacteriophages BPs, Angel and Halo are closely related viruses isolated from Mycobacterium smegmatis, and possess the smallest known mycobacteriophage genomes, 41 901 bp, 42 289 bp and 41 441 bp, respectively. Comparative genome analysis reveals a novel class of ultra-small mobile genetic elements; BPs and Halo each contain an insertion of the proposed mobile elements MPME1 and MPME2, respectively, at different locations, while Angel contains neither. The close similarity of the genomes provides a comparison of the pre- and post-integration sequences, revealing an unusual 6 bp insertion at one end of the element and no target duplication. Nine additional copies of these mobile elements are identified in a variety of different contexts in other mycobacteriophage genomes. In addition, BPs, Angel and Halo have an unusual lysogeny module in which the repressor and integrase genes are closely linked. The attP site is located within the repressor-coding region, such that prophage formation results in expression of a C-terminally truncated, but active, form of the repressor.

In fish, amino acids are food-related important olfactory cues to elicit an attractive response. However, the neural circuit underlying this olfactory behavior is not fully elucidated. In the present study, we applied the Tol2 transposon-mediated gene trap method to dissect the zebrafish olfactory system genetically. Four zebrafish lines (SAGFF27A, SAGFF91B, SAGFF179A, and SAGFF228C) were established in which the modified transcription activator Gal4FF was expressed in distinct subsets of olfactory sensory neurons (OSNs). The OSNs in individual lines projected axons to partially overlapping but mostly different glomeruli in the olfactory bulb (OB). In SAGFF27A, Gal4FF was expressed predominantly in microvillous OSNs innervating the lateral glomerular cluster that corresponded to the amino acid-responsive region in the OB. To clarify the olfactory neural pathway mediating the feeding behavior, we genetically expressed tetanus neurotoxin in the Gal4FF lines to block synaptic transmission in distinct populations of glomeruli and examined their behavioral response to amino acids. The attractive response to amino acids was abolished only in SAGFF27A fish carrying the tetanus neurotoxin transgene. These findings clearly demonstrate the functional significance of the microvillous OSNs innervating the lateral glomerular cluster in the amino acid-mediated feeding behavior of zebrafish. Thus, the integrated approach combining genetic, neuroanatomical, and behavioral methods enables us to elucidate the neural circuit mechanism underlying various olfactory behaviors in adult zebrafish. feeding behavior | Gal4/UAS | neural transmission blockade | olfaction

Despite comprising much of the eukaryotic genome, few transposons are active, and they usually confer no benefit to the host. Through an exaggerated process of genome rearrangement, Oxytricha trifallax destroys 95% of its germline genome during development. This includes the elimination of all transposon DNA. We show that germline-limited transposase genes play key roles in this process of genome-wide DNA excision, which suggests that transposases function in large eukaryotic genomes containing thousands of active transposons. We show that transposase gene expression occurs during germline-soma differentiation and that silencing of transposase by RNA interference reads to abnormal DNA rearrangement in the offspring. This study suggests a new important role in Oxytricha for this large portion of genomic DNA that was previously thought of as junk.

Helitrons are novel transposons discovered by bioinformatic analysis of eukaryotic genome sequences. They are believed to move by rolling circle (RC) replication because their predicted transposases are homologous to those of bacterial RC transposons. We report here evidence of somatic Helitron excision in maize, an unexpected finding suggesting that Helitrons can exhibit an excisive mode of transposition. DOI: 10.1534/genetics.109.101527

Transposition activity in bacteria is generally maintained at a low level. The activity of mobile DNA elements can be controlled by bacterially encoded global regulators. Regulation of transposition of Tn4652 in Pseudomonas putida is one such example. Activation of transposition of Tn4652 in starving bacteria requires the stationary-phase sigma factor RpoS and integration host factor (IHF). IHF plays a dual role in Tn4652 translocation by activating transcription of the transposase gene tnpA of the transposon and facilitating TnpA binding to the inverted repeats of the transposon. Our previous results have indicated that besides IHF some other P. putida-encoded global regulator(s) might bind to the ends of Tn4652 and regulate transposition activity. In this study, employing a DNase I footprint assay we have identified a binding site of P. putida Fis (factor for inversion stimulation) centred 135 bp inside the left end of Tn4652. Our results of gel mobility shift and DNase I footprint studies revealed that Fis out-competes IHF from the left end of Tn4652, thereby abolishing the binding of TnpA. Thus, the results obtained in this study indicate that the transposition of Tn4652 is regulated by the cellular amount of P. putida global regulators Fis and IHF.

Clinical isolates of Pseudomonas aeruginosa that hyperproduce a dark-brown pigment are quite often found in the lungs of chronically infected patients, suggesting that they may have an adaptive advantage in chronic infections. We have screened a library of random transposon insertions in P. aeruginosa. Transposon insertions resulting in the hyperproduction of a dark-brown pigment were found to be located in the hmgA gene, which putatively encodes the enzyme homogentisate-1,2-dioxygenase. Complementation studies indicate that hmgA disruption is responsible for the hyperproduction of pyomelanin in both laboratory and clinical isolates. A relationship between hmgA disruption and adaptation to chronic infection was explored and our results show that the inactivation of hmgA produces a slight reduction of killing ability in an acute murine model of lung infection. On the other hand, it also confers decreased clearance and increased persistence in chronic lung infections. Whether pyomelanin production is the cause of the increased adaptation to chronicity or just a side effect of hmgA inactivation is a question to be studied in future; however, this adaptation is consistent with the higher resistance to oxidative stress conferred in vitro by the pyomelanin pigment. Our results clearly demonstrate that hmgA inactivation leads to a better adaptation to chronic infection, and strongly suggest that this mechanism may be exploited in naturally occurring P. aeruginosa strains.

Early studies of the Bacteroides mobilizable transposon NBU1 established that excision, the first step in NBU1 transfer, requires exposure of the cells to tetracycline. More recently, we found that excision is also associated with growth phase; even after exposure to tetracycline, excision is detectable only after the cells enter late exponential phase. The tetracycline effect is mediated by a two-component regulatory system, RteA and RteB, which is provided in trans by an integrated self-transmissible element, CTnDOT. The rteA and rteB genes are part of a three-gene operon that also contains the tetracycline resistance gene tetQ. We report here that neither transcription nor translation of the tetQ-rteA-rteB operon is affected by growth phase. Moreover, RteA is not required for the growth phase effect, because a mutant form of RteB that does not require phosphorylation by RteA did not make excision independent of growth phase. Two conditions made NBU1 excision independent of growth phase. One was reducing the tetracycline concentration from an inhibitory concentration (1 [micro]g/ml) to a subinhibitory level (0.05 [micro]g/ml). Independence of growth phase also occurred when rteA and rteB were placed under the control of a heterologous maltose-inducible promoter, [P.sub.susA]. Our results suggest that at low concentrations of tetracycline, ribosomes are capable of translating enough RteA and RteB for excision to occur. At higher tetracycline concentrations, however, TetQ is needed to protect enough ribosomes to allow the translation of excision genes, and this protection takes time to develop. Thus, subinhibitory concentrations of tetracycline may increase the probability of gene transfer because, in contrast to inhibitory concentrations, excision can occur at all phases of growth.

The goal of this study was to determine the impact of breeding system and the presence of the transposon Pokey on intraindividual variation in 28S rRNA genes. We PCR-amplified, cloned, and sequenced 1000 nucleotides downstream of the Pokey insertion site in genes with and without insertions from 10 obligately and 10 cyclically parthenogenetic isolates of Daphnia pulex. Variation among genes with Pokey insertions was higher than variation among genes without insertions in both cyclic and obligate isolates. Although the differences were not quite significant (p = 0.06 in both cases), the results suggest that Pokey insertions are likely to inhibit the homogenization of their host genes to some extent. We also observed that the complement of 28S rRNA alleles differed between genes with and without inserts in some isolates, suggesting that a particular inserted gene can persist for substantial periods of time and even spread within the rDNA array, despite the fact that insertions are deleterious. This apparently contradictory pattern can be explained if homogenization of rRNA genes occurs primarily by gene conversion, but copies with Pokey inserts can occasionally increase in frequency within arrays owing to unequal crossing over events that do not originate in the inserted genes themselves. Key words: concerted evolution, Pokey, DNA transposon, parthenogenesis, Daphnia pulex, rDNA, 28S rRNA. Le but de ce travail etait de determiner l'impact du mode de reproduction et de la presence du transposon Pokey sur la variation intra-individuelle parmi les genes codant pour les ARNr 28S. Les auteurs ont amplifie, clone et sequence 1000 nucleotides en aval du site d'insertion de Pokey dans des genes avec ou sans insertion chez 10 isolats parthenogeniques obligatoires et chez 10 isolats parthenogenique cycliques du Daphnia pulex. La variation au sein des genes contenant des insertions Pokey etait plus grande qu'au sein de genes sans insertion tant chez les isolats cycliques qu'obligatoires. Bien que les differences n'etaient pas tout a fait significatives (p = 0,06 dans les deux cas), les resultats suggerent que les insertions Pokey pourraient inhiber l'homogeneisation de leurs genes h6tes jusqu'a un certain point. Les auteurs ont egalement observe que le complement d'alleles d'ARNr 28S differait entre genes avec et sans insertion chez certains isolats, ce qui suggere qu'un gene particulier comprenant une insertion pent persister pour des periodes substantielles de temps et meme se repandre au sein de l'amas d'ADNr, malgre le fait que ces insertions soient deleteres. Ces observations apparemment contradictoires pourraient s'expliquer si l'homogeneisation des genes d'ARNr se produit principalement par conversion, mais que les copies avec des insertions de Pokey peuvent occasionnellement se multiplier au sein des amas par suite d'evenements de recombinaison inegale qui n'ont pas leur origine au sein des genes dotes d'insertions. Mots-cles : evolution concertee, Pokey, transposon a ADN, parthenogenese, Daphnia pulex, ADNr, ARNr 28S. [Traduit par la Redaction], Introduction Eukaryotic ribosomal DNA (rDNA) contains tandem arrays of a repeat unit composed of 3 co-transcribed ribosomal RNA (rRNA) genes (18S, 5.8S, and 28S in metazoans) separated by intergenic spacers [...]