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1.
Mol Brain ; 14(1): 56, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33726803

RESUMO

Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterized by core symptoms of impaired social behavior and communication. Recent studies have suggested that the oxytocin system, which regulates social behavior in mammals, is potentially involved in ASD. Mouse models of ASD provide a useful system for understanding the associations between an impaired oxytocin system and social behavior deficits. However, limited studies have shown the involvement of the oxytocin system in the behavioral phenotypes in mouse models of ASD. We have previously demonstrated that a mouse model that carries the ASD patient-derived de novo mutation in the pogo transposable element derived with zinc finger domain (POGZWT/Q1038R mice), showed ASD-like social behavioral deficits. Here, we have explored whether oxytocin (OXT) administration improves impaired social behavior in POGZWT/Q1038R mice and found that intranasal oxytocin administration effectively restored the impaired social behavior in POGZWT/Q1038R mice. We also found that the expression level of the oxytocin receptor gene (OXTR) was low in POGZWT/Q1038R mice. However, we did not detect significant changes in the number of OXT-expressing neurons between the paraventricular nucleus of POGZWT/Q1038R mice and that of WT mice. A chromatin immunoprecipitation assay revealed that POGZ binds to the promoter region of OXTR and is involved in the transcriptional regulation of OXTR. In summary, our study demonstrate that the pathogenic mutation in the POGZ, a high-confidence ASD gene, impairs the oxytocin system and social behavior in mice, providing insights into the development of oxytocin-based therapeutics for ASD.


Assuntos
Transtorno do Espectro Autista/tratamento farmacológico , Ocitocina/uso terapêutico , Comportamento Social , Transposases/genética , Administração Intranasal , Animais , Transtorno do Espectro Autista/psicologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Humanos , Camundongos , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ocitocina/administração & dosagem , Ocitocina/farmacologia , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Receptores de Ocitocina/biossíntese , Receptores de Ocitocina/genética , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/genética , Transcrição Gênica , Transposases/fisiologia
2.
Int J Mol Sci ; 21(21)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172005

RESUMO

The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast "cut and paste" and "copy and paste" functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transposases/genética , Transposases/metabolismo , Animais , Cromatina/genética , Epigenômica/métodos , Variação Genética/genética , Genômica/métodos , Humanos , Análise de Sequência de DNA/métodos , Transposases/fisiologia
3.
PLoS Genet ; 16(5): e1008681, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463832

RESUMO

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/metabolismo , Transposases/fisiologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Domínio Catalítico/genética , Células Cultivadas , Domesticação , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb/genética , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Células Sf9 , Spodoptera , Transposases/genética
4.
Nucleic Acids Res ; 46(19): 10286-10301, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30239795

RESUMO

Some DNA transposons relocate from one genomic location to another using a mechanism that involves generating double-strand breaks at their transposon ends by forming hairpins on flanking DNA. The same double-strand break mode is employed by the V(D)J recombinase at signal-end/coding-end junctions during the generation of antibody diversity. How flanking hairpins are formed during DNA transposition has remained elusive. Here, we describe several co-crystal structures of the Hermes transposase bound to DNA that mimics the reaction step immediately prior to hairpin formation. Our results reveal a large DNA conformational change between the initial cleavage step and subsequent hairpin formation that changes which strand is acted upon by a single active site. We observed that two factors affect the conformational change: the complement of divalent metal ions bound by the catalytically essential DDE residues, and the identity of the -2 flanking base pair. Our data also provides a mechanistic link between the efficiency of hairpin formation (an A:T basepair is favored at the -2 position) and Hermes' strong target site preference. Furthermore, we have established that the histidine residue within a conserved C/DxxH motif present in many transposase families interacts directly with the scissile phosphate, suggesting a crucial role in catalysis.


Assuntos
Quebras de DNA de Cadeia Dupla , Clivagem do DNA , Eucariotos/enzimologia , Transposases/fisiologia , Animais , Sítios de Ligação , Catálise , Domínio Catalítico , Elementos de DNA Transponíveis , Eucariotos/genética , Eucariotos/metabolismo , Células Eucarióticas/enzimologia , Células Eucarióticas/metabolismo , Humanos , Família Multigênica , Conformação Proteica , Transposases/química , Transposases/genética
5.
Nat Genet ; 49(7): 1005-1014, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28504702

RESUMO

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.


Assuntos
Transformação Celular Neoplásica/genética , Tumor Rabdoide/genética , Transposases/fisiologia , Adulto , Animais , Domínio Catalítico , Linhagem Celular , Criança , Pré-Escolar , Aberrações Cromossômicas , Pontos de Quebra do Cromossomo , Reparo do DNA por Junção de Extremidades/genética , DNA de Neoplasias/genética , Rearranjo Gênico/genética , Genes Supressores de Tumor , Humanos , Lactente , Camundongos , Camundongos Nus , Mutagênese Sítio-Dirigida , Interferência de RNA , Proteínas Recombinantes/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sequências Repetidas Terminais/genética , Transposases/química , Transposases/genética
6.
Cancer Res ; 77(6): 1357-1368, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28108518

RESUMO

RAS genes are mutated in 20% of human tumors, but these mutations are very rare in breast cancer. Here, we used a mouse model to generate tumors upon activation of a mutagenic T2Onc2 transposon via expression of a transposase driven by the keratin K5 promoter in a p53+/- background. These animals mainly developed mammary tumors, most of which had transposon insertions in one of two RASGAP genes, neurofibromin1 (Nf1) and RAS p21 protein activator (Rasa1). Immunohistochemical analysis of a collection of human breast tumors confirmed that low expression of RASA1 is frequent in basal (triple-negative) and estrogen receptor negative tumors. Bioinformatic analysis of human breast tumors in The Cancer Genome Atlas database showed that although RASA1 mutations are rare, allelic loss is frequent, particularly in basal tumors (80%) and in association with TP53 mutation. Inactivation of RASA1 in MCF10A cells resulted in the appearance of a malignant phenotype in the context of mutated p53. Our results suggest that alterations in the Ras pathway due to the loss of negative regulators of RAS may be a common event in basal breast cancer. Cancer Res; 77(6); 1357-68. ©2017 AACR.


Assuntos
Biomarcadores Tumorais/metabolismo , Elementos de DNA Transponíveis/genética , Transposases/fisiologia , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/fisiologia , Proteína p120 Ativadora de GTPase/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Células Cultivadas , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Estadiamento de Neoplasias , Prognóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína p120 Ativadora de GTPase/genética
7.
FEBS J ; 284(11): 1590-1605, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27973733

RESUMO

The adaptive immune system of jawed vertebrates relies on V(D)J recombination as one of the main processes to generate the diverse array of receptors necessary for the recognition of a wide range of pathogens. The DNA cleavage reaction necessary for the assembly of the antigen receptor genes from an array of potential gene segments is mediated by the recombination-activating gene proteins RAG1 and RAG2. The RAG proteins have been proposed to originate from a transposable element (TE) as they share mechanistic and structural similarities with several families of transposases and are themselves capable of mediating transposition. A number of RAG-like proteins and TEs with sequence similarity to RAG1 and RAG2 have been identified, but only recently has their function begun to be characterized, revealing mechanistic links to the vertebrate RAGs. Of particular significance is the discovery of ProtoRAG, a transposon superfamily found in the genome of the basal chordate amphioxus. ProtoRAG has many of the sequence and mechanistic features predicted for the ancestral RAG transposon and is likely to be an evolutionary relative of RAG1 and RAG2. In addition, early observations suggesting that RAG1 is able to mediate V(D)J recombination in the absence of RAG2 have been confirmed, implying independent evolutionary origins for the two RAG genes. Here, recent progress in identifying and characterizing RAG-like proteins and the TEs that encode them is summarized and a refined model for the evolution of V(D)J recombination and the RAG proteins is presented.


Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/fisiologia , Evolução Molecular , Genes RAG-1 , Proteínas de Homeodomínio/fisiologia , Recombinação V(D)J , Vertebrados/imunologia , Animais , Sequência Conservada , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Transferência Genética Horizontal , Humanos , Anfioxos/genética , Anfioxos/imunologia , Modelos Genéticos , Filogenia , Ouriços-do-Mar/genética , Ouriços-do-Mar/imunologia , Estrelas-do-Mar/genética , Estrelas-do-Mar/imunologia , Transposases/genética , Transposases/fisiologia , VDJ Recombinases/genética , VDJ Recombinases/fisiologia , Vertebrados/genética
9.
Nat Genet ; 47(1): 47-56, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25485836

RESUMO

Here we describe a conditional piggyBac transposition system in mice and report the discovery of large sets of new cancer genes through a pancreatic insertional mutagenesis screen. We identify Foxp1 as an oncogenic transcription factor that drives pancreatic cancer invasion and spread in a mouse model and correlates with lymph node metastasis in human patients with pancreatic cancer. The propensity of piggyBac for open chromatin also enabled genome-wide screening for cancer-relevant noncoding DNA, which pinpointed a Cdkn2a cis-regulatory region. Histologically, we observed different tumor subentities and discovered associated genetic events, including Fign insertions in hepatoid pancreatic cancer. Our studies demonstrate the power of genetic screening to discover cancer drivers that are difficult to identify by other approaches to cancer genome analysis, such as downstream targets of commonly mutated human cancer genes. These piggyBac resources are universally applicable in any tissue context and provide unique experimental access to the genetic complexity of cancer.


Assuntos
Transformação Celular Neoplásica/genética , Elementos de DNA Transponíveis/genética , Redes Reguladoras de Genes , Mutagênese Insercional , Neoplasias Pancreáticas/genética , Sequência de Aminoácidos , Animais , Fatores de Transcrição Forkhead/análise , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Genes Sintéticos , Genes p16 , Humanos , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mariposas/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/patologia , ATPases Translocadoras de Prótons/genética , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/análise , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transgenes , Transposases/genética , Transposases/fisiologia
10.
Neuron ; 81(3): 505-20, 2014 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-24507188

RESUMO

We present a method to label and trace the lineage of multiple neural progenitors simultaneously in vertebrate animals via multiaddressable genome-integrative color (MAGIC) markers. We achieve permanent expression of combinatorial labels from new Brainbow transgenes introduced in embryonic neural progenitors with electroporation of transposon vectors. In the mouse forebrain and chicken spinal cord, this approach allows us to track neural progenitor's descent during pre- and postnatal neurogenesis or perinatal gliogenesis in long-term experiments. Color labels delineate cytoarchitecture, resolve spatially intermixed clones, and specify the lineage of astroglial subtypes and adult neural stem cells. Combining colors and subcellular locations provides an expanded marker palette to individualize clones. We show that this approach is also applicable to modulate specific signaling pathways in a mosaic manner while color-coding the status of individual cells regarding induced molecular perturbations. This method opens new avenues for clonal and functional analysis in varied experimental models and contexts.


Assuntos
Encéfalo/citologia , Linhagem da Célula/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Medula Espinal/citologia , Células-Tronco/fisiologia , Fatores Etários , Animais , Animais Recém-Nascidos , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Embrião de Galinha , Colorimetria , Eletroporação , Embrião de Mamíferos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Neurogênese/fisiologia , Medula Espinal/embriologia , Células-Tronco/citologia , Fatores de Tempo , Transposases/fisiologia
12.
PLoS One ; 6(12): e29110, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22216176

RESUMO

Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34(+) cells with DsRed and a hybrid IHK-ß-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased ß-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p=0.05), indicating expression of ß-globin from the integrated SB transgene. IHK-ß-globin mRNA was found in non-erythroid cell types, similar to native ß-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK-ß-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK-ß-globin transgene for gene therapy of sickle cell disease.


Assuntos
Eritrócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Transposases/fisiologia , Globinas beta/metabolismo , Linhagem da Célula , Inativação Gênica , Humanos , Células K562 , RNA Mensageiro/genética , Transgenes , Globinas beta/genética
13.
Mol Ther ; 18(11): 1896-906, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20717103

RESUMO

Sleeping Beauty (SB) transposase enables somatic integration of exogenous DNA in mammalian cells, but potency as a gene transfer vector especially in large mammals has been lacking. Herein, we show that hyperactive transposase system delivered by high-capacity adenoviral vectors (HC-AdVs) can result in somatic integration of a canine factor IX (cFIX) expression-cassette in canine liver, facilitating stabilized transgene expression and persistent haemostatic correction of canine hemophilia B with negligible toxicity. We observed stabilized cFIX expression levels during rapid cell cycling in mice and phenotypic correction of the bleeding diathesis in hemophilia B dogs for up to 960 days. In contrast, systemic administration of an inactive transposase system resulted in rapid loss of transgene expression and transient phenotypic correction. Notably, in dogs a higher viral dose of the active SB transposase system resulted into transient phenotypic correction accompanied by transient increase of liver enzymes. Molecular analysis of liver samples revealed SB-mediated integration and provide evidence that transgene expression was derived mainly from integrated vector forms. Demonstrating that a viral vector system can deliver clinically relevant levels of a therapeutic protein in a large animal model of human disease paves a new path toward the possible cure of genetic diseases.


Assuntos
Modelos Animais de Doenças , Terapia Genética , Hemofilia B/terapia , Transposases/fisiologia , Adenoviridae/genética , Animais , Sequência de Bases , Elementos de DNA Transponíveis/genética , Cães , Fator IX/imunologia , Fator IX/metabolismo , Vetores Genéticos , Hemofilia B/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Homologia de Sequência do Ácido Nucleico , Transgenes/fisiologia
14.
Cancer Genet Cytogenet ; 200(2): 184-90, 2010 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-20620605

RESUMO

Chromosomal translocations are common in leukemia, but little is known about their mechanism. Metnase (also termed SETMAR) is a fusion of a histone methylase and transposase protein that arose specifically in primates. Transposases were thought to be extinct in primates because they would mediate deleterious DNA movement. In primates, Metnase interacts with DNA Ligase IV (Lig IV) and promotes nonhomologous end-joining (NHEJ) DNA repair. We show here that the primate-specific protein Metnase can also enhance NHEJ in murine cells and can also interact with murine Lig IV, indicating that it integrated into the preexisting NHEJ pathway after its development in primates. Significantly, expressing Metnase in murine cells significantly reduces chromosomal translocations. We propose that the fusion of the histone methylase SET domain and the transposase domain in the anthropoid lineage to form primate Metnase promotes accurate intrachromosomal NHEJ and thereby suppresses interchromosomal translocations. Metnase may have been selected for because it has a function opposing transposases and may thus play a key role in suppressing translocations that underlie oncogenicity.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Histona-Lisina N-Metiltransferase/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Translocação Genética , Transposases/fisiologia , Animais , DNA Ligase Dependente de ATP , DNA Ligases/fisiologia , Reparo do DNA , Camundongos , Células NIH 3T3
16.
Semin Cancer Biol ; 20(4): 261-8, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20478384

RESUMO

Significant emphasis has recently been placed on the characterization of the human cancer genome. This effort has been assisted by the development of new DNA sequencing technologies that allow the genomes of individual tumors to be analyzed in much greater detail. However, the genetic complexity of human cancer has complicated the identification of driver mutations among the more abundant passenger mutations found in tumors. Recently, the Sleeping Beauty (SB) transposon system has been engineered to model cancer in mice. SB-induced tumors are produced by transposon insertional mutagenesis, thus the tagged mutations facilitate the identification of novel cancer genes. This review provides a brief summary of the SB system and its use in modeling cancer in mice.


Assuntos
Elementos de DNA Transponíveis/fisiologia , Modelos Animais de Doenças , Estudos de Associação Genética/métodos , Camundongos/genética , Neoplasias/genética , Animais , Genes Neoplásicos , Testes Genéticos/métodos , Humanos , Modelos Biológicos , Neoplasias/diagnóstico , Neoplasias/patologia , Transposases/metabolismo , Transposases/fisiologia
17.
Nat Methods ; 7(6): 451-3, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20418868

RESUMO

We developed a method, MosDEL, to generate targeted knockouts of genes in Caenorhabditis elegans by injection. We generated a double-strand break by mobilizing a Mos1 transposon adjacent to the region to be deleted; the double-stranded break is repaired using injected DNA as a template. Repair can delete up to 25 kb of DNA and simultaneously insert a positive selection marker.


Assuntos
Caenorhabditis elegans/genética , Elementos de DNA Transponíveis/genética , Deleção de Genes , Animais , Hibridização Genômica Comparativa , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Transposases/fisiologia
18.
Crit Rev Biochem Mol Biol ; 45(1): 50-69, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20067338

RESUMO

DNA rearrangements are important in genome function and evolution. Genetic material can be rearranged inadvertently during processes such as DNA repair, or can be moved in a controlled manner by enzymes specifically dedicated to the task. DNA transposases comprise one class of such enzymes. These move DNA segments known as transposons to new locations, without the need for sequence homology between transposon and target site. Several biochemically distinct pathways have evolved for DNA transposition, and genetic and biochemical studies have provided valuable insights into many of these. However, structural information on transposases - particularly with DNA substrates - has proven elusive in most cases. On the other hand, large-scale genome sequencing projects have led to an explosion in the number of annotated prokaryotic and eukaryotic mobile elements. Here, we briefly review biochemical and mechanistic aspects of DNA transposition, and propose that integrating sequence information with structural information using bioinformatics tools such as secondary structure prediction and protein threading can lead not only to an additional level of understanding but possibly also to testable hypotheses regarding transposition mechanisms. Detailed understanding of transposition pathways is a prerequisite for the long-term goal of exploiting DNA transposons as genetic tools and as a basis for genetic medical applications.


Assuntos
Bactérias/genética , Eucariotos/genética , Transposases , Animais , Bactérias/enzimologia , DNA/análise , DNA/genética , DNA/metabolismo , Eucariotos/enzimologia , Rearranjo Gênico , Genoma , Instabilidade Genômica , Humanos , Relação Estrutura-Atividade , Transposases/química , Transposases/fisiologia
19.
Genetica ; 138(3): 301-11, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19763844

RESUMO

Protozoan parasites affect millions of people around the world. Treatment and control of these diseases are complicated partly due to the intricate biology of these organisms. The interactions of species of Plasmodium, Leishmania and trypanosomes with their hosts are mediated by an unusual control of gene expression that is not fully understood. The availability of the genome sequence of these protozoa sets the stage for using more comprehensive, genome-wide strategies to study gene function. Transposons are effective tools for the systematic introduction of genetic alterations and different transposition systems have been adapted to study gene function in these human pathogens. A mariner transposon toolkit for use in vivo or in vitro in Leishmania parasites has been developed and can be used in a variety of applications. These modified mariner elements not only permit the inactivation of genes, but also mediate the rescue of translational gene fusions, bringing a major contribution to the investigation of Leishmania gene function. The piggyBac and Tn5 transposons have also been shown to mobilize across Plasmodium spp. genomes circumventing the current limitations in the genetic manipulation of these organisms.


Assuntos
Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Leishmania/fisiologia , Mutagênese , Animais , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Genoma de Protozoário , Humanos , Plasmodium/fisiologia , Infecções por Protozoários/parasitologia , Transposases/fisiologia , Trypanosoma/fisiologia
20.
Yakugaku Zasshi ; 129(12): 1433-43, 2009 Dec.
Artigo em Japonês | MEDLINE | ID: mdl-19952518

RESUMO

Transposons are mobile genetic elements that move between or within vectors and chromosomes. For the transposition, an enzyme called transposase recognizes transposon-specific terminal inverted repeat sequences (IRs) located on both ends of transposons, and remove them from their original sites and, integrates them into other sites. Because of this feature, transposons containing genes of interest between their two IRs are able to carry the genes from vectors to chromosomes. Transposons are promising systems for chromosomal integration because they can not only integrate exogenous genes efficiently, but also be transfected to a variety of cells or organs using a range of transfection methods. In this review, we focused on the therapeutic application of transposons. A few transposons can integrate transgenes into mammalian chromosomes. They have been used in preclinical studies of gene therapy and cell therapy. In addition, they have recently been used for generation of induced pluripotent stem cells. Transposon-based integrative vector systems have two components. One is the transposon containing transgenes, and the other is the expression cassette of the transposase. Both viral and non-viral vectors have been used to deliver these two components to mammalian cells or organs, and sustained transgene expression has been achieved. Transposon-mediated sustained transgene expression has also produced therapeutic effect in disease models of hereditary and chronic diseases. Although transposon-based integrative vector systems have problems, such as insertional mutagenesis, studies to overcome these problems have been progressing, and these vector systems will become indispensable tools to cure refractory diseases.


Assuntos
Elementos de DNA Transponíveis , Terapia Genética , Vetores Genéticos , Animais , Terapia Baseada em Transplante de Células e Tecidos , Cromossomos/genética , Elementos de DNA Transponíveis/genética , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Sequências Repetidas Invertidas/genética , Mutagênese Insercional , Células-Tronco Pluripotentes , Transfecção/métodos , Transgenes , Transposases/fisiologia
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