http://www.mobilednajournal.com The latest research articles published by Mobile DNA 2012-01-26T00:00:00Z Background: Transposition in the IS3, IS30, IS21 and IS256 insertion sequence(IS) families utilizes an unconventional 2-step pathway. The Step I figure-of-eight intermediate, produced from asymmetric single strand cleavage and joining circularization reactions, is converted into a double stranded minicircle; its junction, the MCJ,[abutted left(IRL)and right(IRR)ends] is the substrate for symmetrical transesterification attacks on target DNA in Step II. This suggests intrinsically different synaptic complexes (SC) for each step. Because transposases of these ISs bind poorly to cognate DNA, comparative biophysical analyses of SC I and SC II have proven elusive. Here we utilize successfully, a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and MCJ substrates, in hydroxyl radical footprinting experiments. Results: IS2 Step I reactions are physically and biochemically asymmetric with IRL the recipient end, lacking donor function. In SC I, different protection patterns of the cleavage domains (CDs) of IRR (extensive in cis) and IRL (selective in trans) at one active catalytic center (the IRR CC), are related to their donor and recipient functions. In SC II, an MCJ substrate showed extensive protection of both CDs, IRL in trans and the abutted IRR CD in cis - the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR, revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages of the abutted CDs bound at the same CC, trigger a conformational change, allowing the IRL CD to complex to its cognate CC - the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding in trans. Conclusions: Different transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor functions of the IRL CD in SC I and SC II and the proposed conformational change in SC II that precedes the symmetrical IRL and IRR donor attacks on target DNA, highlight the differences. http://www.mobilednajournal.com/content/3/1/1 Leslie Lewis Mekbib Astatke Peter Umekubo Shaheen Alvi Robert Saby Jehan Afrose Pedro Oliveira Gabriel Monteiro Duarte Prazeres Mobile DNA 2012, null:1 2012-01-26T00:00:00Z doi:10.1186/1759-8753-3-1 /content/figures/1759-8753-3-1-toc.gif Mobile DNA 1759-8753 ${item.volume} 1 2012-01-26T00:00:00Z PDF Background: The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Results: Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. Conclusions: In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system. http://www.mobilednajournal.com/content/2/1/15 Donald Yergeau Clair Kelley Emin Kuliyev Haiqing Zhu Michelle Johnson Hamlet Amy Sater Dan Wells Paul Mead Mobile DNA 2011, null:15 2011-11-24T00:00:00Z doi:10.1186/1759-8753-2-15 /content/figures/1759-8753-2-15-toc.gif Mobile DNA 1759-8753 ${item.volume} 15 2011-11-24T00:00:00Z XML Background: The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results: A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common to the three dimensional catalytic core motif of the retroviral integrase superfamily. Conclusions: Intractably insoluble transposases, such as the IS2 transposase, prepared by solubilization protocols are often refractory to whole protein structure-function studies. The results described here have validated the use of GFP-tagging and fluorescence-based random mutagenesis in overcoming this limitation at the secondary structure level. http://www.mobilednajournal.com/content/2/1/14 Leslie Lewis Mekbib Astatke Peter Umekubo Shaheen Alvi Robert Saby Jehan Afrose Mobile DNA 2011, null:14 2011-10-27T00:00:00Z doi:10.1186/1759-8753-2-14 /content/figures/1759-8753-2-14-toc.gif Mobile DNA 1759-8753 ${item.volume} 14 2011-10-27T00:00:00Z XML Background: Although humans and chimpanzees have accumulated significant differences in a number of phenotypic traits since diverging from a common ancestor about six million years ago, their genomes are more than 98.5% identical at protein-coding loci. This modest degree of nucleotide divergence is not sufficient to explain the extensive phenotypic differences between the two species. It has been hypothesized that the genetic basis of the phenotypic differences lies at the level of gene regulation and is associated with the extensive insertion and deletion (INDEL) variation between the two species. To test the hypothesis that large INDELs (80 to 12,000 bp) may have contributed significantly to differences in gene regulation between the two species, we categorized human-chimpanzee INDEL variation mapping in or around genes and determined whether this variation is significantly correlated with previously determined differences in gene expression. Results: Extensive, large INDEL variation exists between the human and chimpanzee genomes. This variation is primarily attributable to retrotransposon insertions within the human lineage. There is a significant correlation between differences in gene expression and large human-chimpanzee INDEL variation mapping in genes or in proximity to them. Conclusions: The results presented herein are consistent with the hypothesis that large INDELs, particularly those associated with retrotransposons, have played a significant role in human-chimpanzee regulatory evolution. http://www.mobilednajournal.com/content/2/1/13 Nalini Polavarapu Gaurav Arora Vinay Mittal John McDonald Mobile DNA 2011, null:13 2011-10-25T00:00:00Z doi:10.1186/1759-8753-2-13 /content/figures/1759-8753-2-13-toc.gif Mobile DNA 1759-8753 ${item.volume} 13 2011-10-25T00:00:00Z XML Background: "Domestication" of transposable elements (TEs) led to evolutionary breakthroughs such as the origin of telomerase and the vertebrate adaptive immune system. These breakthroughs were accomplished by the adaptation of molecular functions essential for TEs, such as reverse transcription, DNA cutting and ligation or DNA binding. Cryptons represent a unique class of DNA transposons using tyrosine recombinase (YR) to cut and rejoin the recombining DNA molecules. Cryptons were originally identified in fungi and later in the sea anemone, sea urchin and insects. Results: Herein we report new Cryptons from animals, fungi, oomycetes and diatom, as well as widely conserved genes derived from ancient Crypton domestication events. Phylogenetic analysis based on the YR sequences supports four deep divisions of Crypton elements. We found that the domain of unknown function 3504 (DUF3504) in eukaryotes is derived from Crypton YR. DUF3504 is similar to YR but lacks most of the residues of the catalytic tetrad (R-H-R-Y). Genes containing the DUF3504 domain are potassium channel tetramerization domain containing 1 (KCTD1), KIAA1958, zinc finger MYM type 2 (ZMYM2), ZMYM3, ZMYM4, glutamine-rich protein 1 (QRICH1) and "without children" (WOC). The DUF3504 genes are highly conserved and are found in almost all jawed vertebrates. The sequence, domain structure, intron positions and synteny blocks support the view that ZMYM2, ZMYM3, ZMYM4, and possibly QRICH1, were derived from WOC through two rounds of genome duplication in early vertebrate evolution. WOC is observed widely among bilaterians. There could be four independent events of Crypton domestication, and one of them, generating WOC/ZMYM, predated the birth of bilaterian animals. This is the third-oldest domestication event known to date, following the domestication generating telomerase reverse transcriptase (TERT) and Prp8. Many Crypton-derived genes are transcriptional regulators with additional DNA-binding domains, and the acquisition of the DUF3504 domain could have added new regulatory pathways via protein-DNA or protein-protein interactions. Conclusions: Cryptons have contributed to animal evolution through domestication of their YR sequences. The DUF3504 domains are domesticated YRs of animal Crypton elements. http://www.mobilednajournal.com/content/2/1/12 Kenji Kojima Jerzy Jurka Mobile DNA 2011, null:12 2011-10-19T00:00:00Z doi:10.1186/1759-8753-2-12 /content/figures/1759-8753-2-12-toc.gif Mobile DNA 1759-8753 ${item.volume} 12 2011-10-19T00:00:00Z XML Background: R2 retrotransposable elements exclusively insert in the 28S rRNA genes of their host. Their RNA transcripts are produced by self-processing from a 28S R2 cotranscript. Because full-length R2 transcripts are found in most tissues of R2-active animals, we tested whether new R2 insertions occurred in somatic tissues even though such events would be an evolutionary dead end.FindingsPCR assays were used to identify somatic R2 insertions in isolated adult tissues and larval imaginal discs of Drosophila simulans. R2 somatic mosaics were detected encompassing cells from individual tissues as well as tissues from multiple body segments. The somatic insertions had 5' junction sequences characteristic of germline insertions suggesting they represented authentic retrotransposition events. Conclusions: Body segments are specified early in Drosophila development, thus the detection of the same somatic insertion in cells from multiple tissues suggested that the R2 retrotransposition events had occurred before the blastoderm stage of Drosophila development. R2 activity at this stage, when embryonic nuclei are rapidly dividing in a common cytoplasm, suggests that some retrotransposition events appearing as germline events may correspond to germline mosaicism. http://www.mobilednajournal.com/content/2/1/11 Michael Eickbush Thomas Eickbush Mobile DNA 2011, null:11 2011-09-29T00:00:00Z doi:10.1186/1759-8753-2-11 Mobile DNA retransposition in somatic cells As evidence for the insertion of mobile DNA elements in somatic cells accumulates, somatic retrotransposition in the early development of Drosophila raises many questions as to the biological importance of such events. /content/figures/1759-8753-2-11-toc.gif Mobile DNA 1759-8753 ${item.volume} 11 2011-09-29T00:00:00Z XML Background: The human genome contains approximately one million Alu elements which comprise more than 10% of human DNA by mass. Alu elements possess direction, and are distributed almost equally in positive and negative strand orientations throughout the genome. Previously, it has been shown that closely spaced Alu pairs in opposing orientation (inverted pairs) are found less frequently than Alu pairs having the same orientation (direct pairs). However, this imbalance has only been investigated for Alu pairs separated by 650 or fewer base pairs (bp) in a study conducted prior to the completion of the draft human genome sequence. Results: We performed a comprehensive analysis of all (> 800,000) full-length Alu elements in the human genome. This large sample size permits detection of small differences in the ratio between inverted and direct Alu pairs (I:D). We have discovered a significant depression in the full-length Alu pair I:D ratio that extends to repeat pairs separated by ≤ 350,000 bp. Within this imbalance bubble (those Alu pairs separated by ≤ 350,000 bp), direct pairs outnumber inverted pairs. Using PCR, we experimentally verified several examples of inverted Alu pair exclusions that were caused by deletions. Conclusions: Over 50 million full-length Alu pairs reside within the I:D imbalance bubble. Their collective impact may represent one source of Alu element-related human genomic instability that has not been previously characterized. http://www.mobilednajournal.com/content/2/1/10 George Cook Miriam Konkel James Major Jerilyn Walker Kyudong Han Mark Batzer Mobile DNA 2011, null:10 2011-09-23T00:00:00Z doi:10.1186/1759-8753-2-10 /content/figures/1759-8753-2-10-toc.gif Mobile DNA 1759-8753 ${item.volume} 10 2011-09-23T00:00:00Z XML Background: Determining the mechanisms by which transposable elements move within a genome increases our understanding of how they can shape genome evolution. Class 2 transposable elements transpose via a 'cut-and-paste' mechanism mediated by a transposase that binds to sites at or near the ends of the transposon. Herves is a member of the hAT superfamily of class 2 transposons and was isolated from Anopheles gambiae, a medically important mosquito species that is the major vector of malaria in sub-Saharan Africa. Herves is transpositionally active and intact copies of it are found in field populations of A gambiae. In this study we report the binding activities of the Herves transposase to the sequences at the ends of the Herves transposon and compare these to other sequences recognized by hAT transposases isolated from other organisms. Results: We identified the specific DNA-binding sites of the Herves transposase. Active Herves transposase was purified using an Escherichia coli expression system and bound in a site-specific manner to the subterminal and terminal sequences of the left and right ends of the element, respectively, and also interacted with the right but not the left terminal inverted repeat. We identified a common subterminal DNA-binding motif (CG/AATTCAT) that is critical and sufficient for Herves transposase binding. Conclusions: The Herves transposase binds specifically to a short motif located at both ends of the transposon but shows differential binding with respect to the left and right terminal inverted repeats. Despite similarities in the overall structures of hAT transposases, the regions to which they bind in their respective transposons differ in sequence ensuring the specificity of these enzymes to their respective transposon. The asymmetry with which the Herves terminal inverted repeats are bound by the transposase may indicate that these differ in their interactions with the enzyme. http://www.mobilednajournal.com/content/2/1/9 Amandeep Kahlon Robert Hice David O'Brochta Peter Atkinson Mobile DNA 2011, null:9 2011-06-20T00:00:00Z doi:10.1186/1759-8753-2-9 /content/figures/1759-8753-2-9-toc.gif Mobile DNA 1759-8753 ${item.volume} 9 2011-06-20T00:00:00Z XML Transposable elements (TEs) are increasingly being recognized as powerful facilitators of evolution. We propose the TE-Thrust hypothesis to encompass TE-facilitated processes by which genomes self-engineer coding, regulatory, karyotypic or other genetic changes. Although TEs are occasionally harmful to some individuals, genomic dynamism caused by TEs can be very beneficial to lineages. This can result in differential survival and differential fecundity of lineages. Lineages with an abundant and suitable repertoire of TEs have enhanced evolutionary potential and, if all else is equal, tend to be fecund, resulting in species-rich adaptive radiations, and/or they tend to undergo major evolutionary transitions. Many other mechanisms of genomic change are also important in evolution, and whether the evolutionary potential of TE-Thrust is realized is heavily dependent on environmental and ecological factors. The large contribution of TEs to evolutionary innovation is particularly well documented in the primate lineage. In this paper, we review numerous cases of beneficial TE-caused modifications to the genomes of higher primates, which strongly support our TE-Thrust hypothesis. http://www.mobilednajournal.com/content/2/1/8 Keith Oliver Wayne Greene Mobile DNA 2011, null:8 2011-05-31T00:00:00Z doi:10.1186/1759-8753-2-8 /content/figures/1759-8753-2-8-toc.gif Mobile DNA 1759-8753 ${item.volume} 8 2011-05-31T00:00:00Z XML Background: Endogenous retroviruses (ERVs) and ERV-like sequences comprise 8% of the human genome. A hitherto unknown proportion of ERV loci are transcribed and thus contribute to the human transcriptome. A small proportion of these loci encode functional proteins. As the role of ERVs in normal and diseased biological processes is not yet established, transcribed ERV loci are of particular interest. As more transcribed ERV loci are likely to be identified in the near future, the development of a systematic nomenclature is important to ensure that all information on each locus can be easily retrieved. Results: Here we present a revised nomenclature of transcribed human endogenous retroviral loci that sorts loci into groups based on Repbase classifications. Each symbol is of the format ERV + group symbol + unique number. Group symbols are based on a mixture of Repbase designations and well-supported symbols used in the literature. The presented guidelines will allow newly identified loci to be easily incorporated into the scheme. Conclusions: The naming system will be employed by the HUGO Gene Nomenclature Committee for naming transcribed human ERV loci. We hope that the system will contribute to clarifying a certain aspect of a sometimes confusing nomenclature for human endogenous retroviruses. The presented system may also be employed for naming transcribed loci of human non-ERV repeat loci. http://www.mobilednajournal.com/content/2/1/7 Jens Mayer Jonas Blomberg Ruth Seal Mobile DNA 2011, null:7 2011-05-04T00:00:00Z doi:10.1186/1759-8753-2-7 /content/figures/1759-8753-2-7-toc.gif Mobile DNA 1759-8753 ${item.volume} 7 2011-05-04T00:00:00Z XML