http://www.mobilednajournal.com The latest research articles published by Mobile DNA 2010-07-23T00:00:00Z This is an RSS newsfeed from BioMed Central It is intended to be used with an RSS reader. For more information about RSS newsfeeds from BioMed Central, visit http://www.biomedcentral.com/info/about/rss/ The bacterial transposon Tn7 is distinguished by its ability to recognize a specific site called attTn7, and insert just downstream of the highly conserved chromosomal glmS gene. TnsD is one of four transposon-encoded polypeptides (TnsABC+D) required for site-specific insertion of Tn7 into attTn7, and is the target site-selector that binds to a highly conserved sequence in the end of the glmS protein coding region. In this study, we identified important nucleotides within this region that are crucial for TnsD-attTn7 interaction. We also probed the regions of TnsD that interact with attTn7 and found that there are important DNA-binding determinants throughout the entire length of the protein, including an amino-terminal CCCH zinc-finger motif. A key role of TnsD is to recruit the non-sequence specific DNA-binding protein TnsC to attTn7; TnsC also interacts with and controls both the TnsA and TnsB subunits of the Tn7 transposase. TnsC stimulates the binding of TnsD to attTn7 in vivo, and TnsCD and TnsD can also interact in the absence of DNA and localize their interaction domains to the N-terminal region of each protein. http://www.mobilednajournal.com/content/1/1/18 Rupak Mitra Gregory McKenzie Liang Yi Cherline Lee Nancy Craig Mobile DNA 2010, 1:18 2010-07-23T00:00:00Z doi:10.1186/1759-8753-1-18 Mobile DNA 1759-8753 1 18 2010-07-23T00:00:00Z PDF Background: Long interspersed nuclear element-1 (LINE-1 or L1) is a dominant repetitive sequence in the human genome. Besides mediating its own retrotransposition, L1 can mobilize Alu and messenger RNA (mRNA) in trans, and probably also SVA and non-coding RNA. The structures of L1 copies and trans-mobilized retrocopies are variable and can be classified into three categories: full-length; 5'-truncated; and 5'-inverted insertions. These structures may be generated by different 5' integration mechanisms. Results: In this study, a method to correctly characterize insertions with short target site duplications (TSDs) is developed and extranucleotides, TSDs and microhomologies (MHs) at junctions were analysed for the three types of insertions. Only 5'-truncated L1 insertions were found to be associated with short TSDs. Both full-length and 5'-truncated retrotransposed sequences in trans, including Alu, SVA and mRNA retrocopies and also full-length and 5'-inverted L1, were not associated with short TSDs, indicating the difference of 5' attachment between retrotransposition in cis and retrotransposition in trans. Target sequence analysis suggested that short TSDs were generated in an L1 endonuclease-dependent manner. The MHs were longer for 5'-inverted L1 than for 5'-truncated L1, indicating less dependence on annealing in 5'-truncated L1 insertions. Conclusions: The results suggest that insertions flanked by short TSDs occur more often coupled with the insertion of 5'-truncated L1 than with those of other types of insertions in vivo. The method used in this study can be used to characterize elements without any apparent boundary structures. http://www.mobilednajournal.com/content/1/1/17 Kenji Kojima Mobile DNA 2010, 1:17 2010-07-08T00:00:00Z doi:10.1186/1759-8753-1-17 Mobile DNA 1759-8753 1 17 2010-07-08T00:00:00Z XML Tight regulation of transposition activity is essential to limit damage transposons may cause by generating potentially lethal DNA rearrangements. Assembly of a bona fide protein-DNA complex, the transpososome, within which transposition is catalysed, is a crucial checkpoint in this regulation. In the case of IS911, a member of the large IS3 bacterial insertion sequence family, the transpososome (synaptic complex A; SCA) is composed of the right and left inverted repeated DNA sequences (IRR and IRL) bridged by the transposase, OrfAB (the IS911-encoded enzyme that catalyses transposition). To characterise further this important protein-DNA complex in vitro, we used different tagged and/or truncated transposase forms and analysed their interaction with IS911 ends using gel electrophoresis. Our results allow us to propose a model in which SCA is assembled with a dimeric form of the transposase. Furthermore, we present atomic force microscopy results showing that the terminal inverted repeat sequences are probably assembled in a parallel configuration within the SCA. These results represent the first step in the structural description of the IS911 transpososome, and are discussed in comparison with the very few other transpososome examples described in the literature. http://www.mobilednajournal.com/content/1/1/16 Philippe Rousseau Catherine Tardin Nathalie Toulou Laurence Salome Mick Chandler Mobile DNA 2010, 1:16 2010-06-16T00:00:00Z doi:10.1186/1759-8753-1-16 Mobile DNA 1759-8753 1 16 2010-06-16T00:00:00Z XML Non-long terminal repeat (non-LTR) retrotransposons are present in most eukaryotic genomes. In some species, such as humans, these elements are the most abundant genome sequence and continue to replicate to this day, creating a source of endogenous mutations and potential genotoxic stress. This review will provide a general outline of the replicative cycle of non-LTR retrotransposons. Recent findings regarding the host regulation of non-LTR retrotransposons will be summarized. Finally, future directions of interest will be discussed. http://www.mobilednajournal.com/content/1/1/15 Jeffrey Han Mobile DNA 2010, 1:15 2010-05-12T00:00:00Z doi:10.1186/1759-8753-1-15 Mobile DNA 1759-8753 1 15 2010-05-12T00:00:00Z XML Background: The yeast retrotransposon Ty3 forms stable virus-like particles. Gag3, the major structural protein, is composed of capsid, spacer and nucleocapsid domains. The capsid domain of Gag3 was previously modeled as a structure similar to retrovirus capsid.FindingsTwo-hybrid analysis was used to understand the interactions that contribute to particle assembly. Gag3 interacted with itself as predicted based on its role as the major structural protein. The N-terminal subdomain (NTD) of the capsid was able to interact with itself and with the C-terminal subdomain (CTD) of the capsid, but interacted less well with intact Gag3. Mutations previously shown to block particle assembly disrupted Gag3 interactions more than subdomain interactions. Conclusions: The findings that the NTD interacts with itself and with the CTD are consistent with previous modeling and a role similar to that of the capsid in retrovirus particle structure. These results are consistent with a model in which the Gag3-Gag3 interactions that initiate assembly differ from the subdomain interactions that potentially underlie particle stability. http://www.mobilednajournal.com/content/1/1/14 Min Zhang Liza Larsen Becky Irwin Virginia Bilanchone Suzanne Sandmeyer Mobile DNA 2010, 1:14 2010-05-05T00:00:00Z doi:10.1186/1759-8753-1-14 Mobile DNA 1759-8753 1 14 2010-05-05T00:00:00Z XML Background: The ends of chromosomes, termed telomeres consist of repetitive DNA. The telomeric sequences shorten with cell division and, when telomeres are critically abbreviated, cells stop proliferating. However, in cancer cells, by the expression of telomerase which elongates telomeres, the cells can continue proliferating. Many approaches for telomere shortening have been pursued in the past, but to our knowledge, cutting telomeres in vivo has not so far been demonstrated. In addition, there is lack of information on the cellular effects of telomere shortening in human cells. Results: Here, we created novel chimeric endonucleases to cut telomeres by fusing the endonuclease domain (TRAS1EN) of the silkworm's telomere specific non-long terminal repeat retrotransposon TRAS1 to the human telomere-binding protein, TRF1. An in vitro assay demonstrated that the TRAS1EN-TRF1 chimeric endonucleases (T-EN and EN-T) cut the human (TTAGGG)n repeats specifically. The concentration of TRAS1EN-TRF1 chimeric endonucleases necessary for the cleavage of (TTAGGG)n repeats was about 40-fold lower than that of TRAS1EN alone. When TRAS1EN-TRF1 endonucleases were introduced into human U2OS cancer cells using adenovirus vectors, the enzymes localized at telomeres of nuclei, cleaved and shortened the telomeric DNA by double-strand breaks. When human U2OS and HFL-1 fibroblast cells were infected with EN-T recombinant adenovirus, their cellular proliferation was suppressed for about 2 weeks after infection. In contrast, the TRAS1EN mutant (H258A) chimeric endonuclease fused with TRF1 (ENmut-T) did not show the suppression effect. The EN-T recombinant adenovirus induced telomere shortening in U2OS cells, activated the p53-dependent pathway and caused the senescence associated cellular responses, while the ENmut-T construct did not show such effects. Conclusions: A novel TRAS1EN-TRF1 chimeric endonuclease (EN-T) cuts the human telomeric repeats (TTAGGG)n specifically in vitro and in vivo. Thus, the chimeric endonuclease which is expressed from an adenoviral vector can suppress cell proliferation of cancer cells. http://www.mobilednajournal.com/content/1/1/13 Kazutoshi Yoshitake Hideyuki Aoyagi Haruhiko Fujiwara Mobile DNA 2010, 1:13 2010-04-01T00:00:00Z doi:10.1186/1759-8753-1-13 Mobile DNA 1759-8753 1 13 2010-04-01T00:00:00Z XML Background: Rotifers of the class Bdelloidea are microscopic freshwater invertebrates best known for: their capacity for anhydrobiosis; the lack of males and meiosis; and for the ability to capture genes from other non-metazoan species. Although genetic exchange between these animals might take place by non-canonical means, the overall lack of meiosis and syngamy should greatly impair the ability of transposable elements (TEs) to spread in bdelloid populations. Previous studies demonstrated that bdelloid chromosome ends, in contrast to gene-rich regions, harbour various kinds of TEs, including specialized telomere-associated retroelements, as well as DNA TEs and retrovirus-like retrotransposons which are prone to horizontal transmission. Vertically-transmitted retrotransposons have not previously been reported in bdelloids and their identification and studies of the patterns of their distribution and evolution could help in the understanding of the high degree of TE compartmentalization within bdelloid genomes. Results: We identified and characterized a non-long terminal repeat (LTR) retrotransposon residing primarily in subtelomeric regions of the genome in the bdelloid rotifer Adineta vaga. Contrary to the currently prevailing views on the mode of proliferation of non-LTR retrotransposons, which results in frequent formation of 5'-truncated ('dead-on-arrival') copies due to the premature disengagement of the element-encoded reverse transcriptase from its template, this non-LTR element, Hebe, is represented only by non-5'-truncated copies. Most of these copies, however, were subject to internal deletions associated with microhomologies, a hallmark of non-homologous end-joining events. Conclusions: The non-LTR retrotransposon Hebe from the bdelloid rotifer A. vaga was found to undergo frequent microhomology-associated deletions, rather than 5'-terminal truncations characteristic of this class of retrotransposons, and to exhibit preference for telomeric localization. These findings represent the first example of a vertically transmitted putatively deleterious TE in bdelloids, and may indicate the involvement of microhomology-mediated non-homologous end-joining in desiccation-induced double-strand break repair at the genome periphery. http://www.mobilednajournal.com/content/1/1/12 Eugene Gladyshev Irina Arkhipova Mobile DNA 2010, 1:12 2010-04-01T00:00:00Z doi:10.1186/1759-8753-1-12 Mobile DNA 1759-8753 1 12 2010-04-01T00:00:00Z XML Background: Extrachomosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms and was detected in every organism tested, including in humans. A two-dimensional gel electrophoresis facilitates the detection of eccDNA in preparations of genomic DNA. Using this technique we have previously demonstrated that most of eccDNA consists of exact multiples of chromosomal tandemly repeated DNA, including both coding genes and satellite DNA. Results: Here we report the occurrence of eccDNA in every tested human cell line. It has heterogeneous mass ranging from less than 2 kb to over 20 kb. We describe eccDNA homologous to human alpha satellite and the SstI mega satellite. Moreover, we show, for the first time, circular multimers of the human 5S ribosomal DNA (rDNA), similar to previous findings in Drosophila and plants. We further demonstrate structures that correspond to intermediates of rolling circle replication, which emerge from the circular multimers of 5S rDNA and SstI satellite. Conclusions: These findings, and previous reports, support the general notion that every chromosomal tandem repeat is prone to generate eccDNA in eukryoric organisms including humans. They suggest the possible involvement of eccDNA in the length variability observed in arrays of tandem repeats. The implications of eccDNA on genome biology may include mechanisms of centromere evolution, concerted evolution and homogenization of tandem repeats and genomic plasticity. http://www.mobilednajournal.com/content/1/1/11 Sarit Cohen Neta Agmon Olga Sobol Daniel Segal Mobile DNA 2010, 1:11 2010-03-08T00:00:00Z doi:10.1186/1759-8753-1-11 Mobile DNA 1759-8753 1 11 2010-03-08T00:00:00Z XML Background: Sadhu elements are non-autonomous retroposons first recognized in Arabidopsis thaliana. There is a wide degree of divergence among different elements, suggesting that these sequences are ancient in origin. Here we report the results of several lines of investigation into the genomic organization and evolutionary history of this element family. Results: We present a classification scheme for Sadhu elements in A. thaliana, describing derivative elements related to the full-length elements we reported previously. We characterized Sadhu5 elements in a set of A. thaliana strains in order to trace the history of radiation in this subfamily. Sequences surrounding the target sites of different Sadhu insertions are consistent with mobilization by LINE retroelements. Finally, we identified Sadhu elements grouping into distinct subfamilies in two related species, Arabidopsis arenosa and Arabidopsis lyrata. Conclusions: Our analyses suggest that the Sadhu retroelement family has undergone target primed reverse transcription-driven retrotransposition during the divergence of different A. thaliana strains. In addition, Sadhu elements can be found at moderate copy number in three distinct Arabidopsis species, indicating that the evolutionary history of these sequences can be traced back at least several millions of years. http://www.mobilednajournal.com/content/1/1/10 Sanjida Rangwala Eric Richards Mobile DNA 2010, 1:10 2010-03-01T00:00:00Z doi:10.1186/1759-8753-1-10 Mobile DNA 1759-8753 1 10 2010-03-01T00:00:00Z XML Background: Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. Results: This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. Conclusions: This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development. http://www.mobilednajournal.com/content/1/1/9 Gina Scott Erika de Wynter Graham Cook Mobile DNA 2010, 1:9 2010-03-01T00:00:00Z doi:10.1186/1759-8753-1-9 Mobile DNA 1759-8753 1 9 2010-03-01T00:00:00Z XML