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1.
Nucleic Acids Res ; 45(1): 311-326, 2017 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-27913727

RESUMO

The functional relevance of the inverted repeat structure (IR/DR) in a subgroup of the Tc1/mariner superfamily of transposons has been enigmatic. In contrast to mariner transposition, where a topological filter suppresses single-ended reactions, the IR/DR orchestrates a regulatory mechanism to enforce synapsis of the transposon ends before cleavage by the transposase occurs. This ordered assembly process shepherds primary transposase binding to the inner 12DRs (where cleavage does not occur), followed by capture of the 12DR of the other transposon end. This extra layer of regulation suppresses aberrant, potentially genotoxic recombination activities, and the mobilization of internally deleted copies in the IR/DR subgroup, including Sleeping Beauty (SB). In contrast, internally deleted sequences (MITEs) are preferred substrates of mariner transposition, and this process is associated with the emergence of Hsmar1-derived miRNA genes in the human genome. Translating IR/DR regulation to in vitro evolution yielded an SB transposon version with optimized substrate recognition (pT4). The ends of SB transposons excised by a K248A excision+/integration- transposase variant are processed by hairpin resolution, representing a link between phylogenetically, and mechanistically different recombination reactions, such as V(D)J recombination and transposition. Such variants generated by random mutation might stabilize transposon-host interactions or prepare the transposon for a horizontal transfer.


Assuntos
Reparo do DNA por Junção de Extremidades , Elementos de DNA Transponíveis , Reparo de DNA por Recombinação , Transposases/genética , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Sequências Repetidas Invertidas , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transposases/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Nat Commun ; 7: 10716, 2016 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-26931494

RESUMO

Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition. A hairpin close to the 3'-end of the transposon functions as a transposition terminator. However, the 3'-end can be bypassed by the transposase, resulting in transduction of flanking sequences to new genomic locations. Helraiser transposition generates covalently closed circular intermediates, suggestive of a replicative transposition mechanism, which provides a powerful means to disseminate captured transcriptional regulatory signals across the genome. Indeed, we document the generation of novel transcripts by Helitron promoter capture both experimentally and by transcriptome analysis in bats. Our results provide mechanistic insight into Helitron transposition, and its impact on diversification of gene function by genome shuffling.


Assuntos
Quirópteros/genética , Elementos de DNA Transponíveis/genética , Variação Genética , Genoma , Animais , Células HeLa , Humanos
3.
PLoS One ; 6(8): e23573, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21897845

RESUMO

Genetic engineering can expand the utility of pigs for modeling human diseases, and for developing advanced therapeutic approaches. However, the inefficient production of transgenic pigs represents a technological bottleneck. Here, we assessed the hyperactive Sleeping Beauty (SB100X) transposon system for enzyme-catalyzed transgene integration into the embryonic porcine genome. The components of the transposon vector system were microinjected as circular plasmids into the cytoplasm of porcine zygotes, resulting in high frequencies of transgenic fetuses and piglets. The transgenic animals showed normal development and persistent reporter gene expression for >12 months. Molecular hallmarks of transposition were confirmed by analysis of 25 genomic insertion sites. We demonstrate germ-line transmission, segregation of individual transposons, and continued, copy number-dependent transgene expression in F1-offspring. In addition, we demonstrate target-selected gene insertion into transposon-tagged genomic loci by Cre-loxP-based cassette exchange in somatic cells followed by nuclear transfer. Transposase-catalyzed transgenesis in a large mammalian species expands the arsenal of transgenic technologies for use in domestic animals and will facilitate the development of large animal models for human diseases.


Assuntos
Elementos de DNA Transponíveis/genética , Técnicas de Transferência de Genes , Genoma/genética , Espermatozoides/metabolismo , Suínos/genética , Transgenes/genética , Zigoto/metabolismo , Animais , Animais Geneticamente Modificados , Citoplasma/genética , Feminino , Injeções , Masculino , Plasmídeos/genética , Plasmídeos/metabolismo , Transposases/genética , Transposases/metabolismo , Zigoto/citologia
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