RESUMO
Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.
Assuntos
Elementos de DNA Transponíveis , Moscas Domésticas/enzimologia , Transposases/química , Animais , Sequência de Bases , Cristalografia por Raios X , Dimerização , Moscas Domésticas/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transposases/genética , Transposases/metabolismoRESUMO
OBJECTIVES: Noninvasive ventilation is widely used to avoid tracheal intubation in critically ill children. The objective of this study was to assess whether noninvasive ventilation failure was associated with severe tracheal intubation-associated events and severe oxygen desaturation during tracheal intubation. DESIGN: Prospective multicenter cohort study of consecutive intubated patients using the National Emergency Airway Registry for Children registry. SETTING: Thirteen PICUs (in 12 institutions) in the United States and Canada. PATIENTS: All patients undergoing tracheal intubation in participating sites were included. Noninvasive ventilation failure group included children with any use of high-flow nasal cannula, continuous positive airway pressure, or bilevel noninvasive ventilation in the 6 hours prior to tracheal intubation. Primary tracheal intubation group included children without exposure to noninvasive ventilation within 6 hours before tracheal intubation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Severe tracheal intubation-associated events (cardiac arrest, esophageal intubation with delayed recognition, emesis with aspiration, hypotension requiring intervention, laryngospasm, pneumothorax, pneumomediastinum) and severe oxygen desaturation (< 70%) were recorded prospectively. The study included 956 tracheal intubation encounters; 424 tracheal intubations (44%) occurred after noninvasive ventilation failure, with a median of 13 hours (interquartile range, 4-38 hr) of noninvasive ventilation. Noninvasive ventilation failure group included more infants (47% vs 33%; p < 0.001) and patients with a respiratory diagnosis (56% vs 30%; p < 0.001). Noninvasive ventilation failure was not associated with severe tracheal intubation-associated events (5% vs 5% without noninvasive ventilation; p = 0.96) but was associated with severe desaturation (15% vs 9% without noninvasive ventilation; p = 0.005). After controlling for baseline differences, noninvasive ventilation failure was not independently associated with severe tracheal intubation-associated events (p = 0.35) or severe desaturation (p = 0.08). In the noninvasive ventilation failure group, higher FIO2 before tracheal intubation (≥ 70%) was associated with severe tracheal intubation-associated events. CONCLUSIONS: Critically ill children are frequently exposed to noninvasive ventilation before intubation. Noninvasive ventilation failure was not independently associated with severe tracheal intubation-associated events or severe oxygen desaturation compared to primary tracheal intubation.
Assuntos
Estado Terminal , Unidades de Terapia Intensiva Pediátrica/estatística & dados numéricos , Intubação Intratraqueal/efeitos adversos , Ventilação não Invasiva/efeitos adversos , Oxigênio/sangue , Adolescente , Criança , Pré-Escolar , Pressão Positiva Contínua nas Vias Aéreas , Humanos , Lactente , Estudos Prospectivos , Adulto JovemRESUMO
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/genéticaRESUMO
The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.
Assuntos
Proteínas de Ligação a DNA/química , Transposases/química , Sequência de Bases , DNA/química , DNA/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Transposases/genética , Transposases/metabolismo , Zinco/química , Dedos de ZincoRESUMO
OBJECTIVES: To evaluate if the use of apneic oxygenation during tracheal intubation in children is feasible and would decrease the occurrence of oxygen desaturation. DESIGN: Prospective pre/post observational study. SETTING: A large single-center noncardiac PICU in North America. PATIENTS: All patients less than 18 years old who underwent primary tracheal intubation from August 1, 2014, to September 30, 2018. INTERVENTIONS: Implementation of apneic oxygenation for all primary tracheal intubation as quality improvement. MEASUREMENTS AND MAIN RESULTS: Total of 1,373 tracheal intubations (661 preimplementation and 712 postimplementation) took place during study period. Within 2 months, apneic oxygenation use reached to predefined adherence threshold (> 80% of primary tracheal intubations) after implementation and sustained at greater than 70% level throughout the postimplementation. Between the preimplementation and postimplementation, no significant differences were observed in patient demographics, difficult airway features, or providers. Respiratory and procedural indications were more common during preintervention. Video laryngoscopy devices were used more often during the postimplementation (pre 5% vs post 75%; p < 0.001). Moderate oxygen desaturation less than 80% were observed in fewer tracheal intubations after apneic oxygenation implementation (pre 15.4% vs post 11.8%; p = 0.049); severe oxygen desaturation less than 70% was also observed in fewer tracheal intubations after implementation (pre 10.4% vs post 7.2%; p = 0.032). Hemodynamic tracheal intubation associated events (i.e., cardiac arrests, hypotension, dysrhythmia) were unchanged (pre 3.2% vs post 2.0%; p = 0.155). Multivariable analyses showed apneic oxygenation implementation was significantly associated with a decrease in moderate desaturation less than 80% (adjusted odds ratio, 0.55; 95% CI, 0.34-0.88) and with severe desaturation less than 70% (adjusted odds ratio, 0.54; 95% CI, 0.31-0.96) while adjusting for tracheal intubation indications and device. CONCLUSIONS: Implementation of apneic oxygenation in PICU was feasible, and was associated with significant reduction in moderate and severe oxygen desaturation. Use of apneic oxygenation should be considered when intubating critically ill children.
Assuntos
Unidades de Terapia Intensiva Pediátrica/organização & administração , Intubação Intratraqueal/métodos , Melhoria de Qualidade/organização & administração , Respiração Artificial/métodos , Centros Médicos Acadêmicos , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Intubação Intratraqueal/efeitos adversos , Masculino , Oxigênio/sangue , Estudos Prospectivos , Adulto JovemRESUMO
RATIONALE: Many in-hospital cardiac arrests are precipitated by hypotension, often associated with systemic inflammation. These patients are less likely to be successfully resuscitated, and novel approaches to their treatment are needed. OBJECTIVES: To determine if the addition of inhaled nitric oxide (iNO) to hemodynamic-directed cardiopulmonary resuscitation (HD-CPR) would improve short-term survival from cardiac arrest associated with shock and systemic inflammation. METHODS: In 3-month-old swine (n = 21), LPS was intravenously infused, inducing systemic hypotension. Ventricular fibrillation was induced, and animals were randomized to blinded treatment with either: 1) HD-CPR with iNO, or 2) HD-CPR without iNO. During HD-CPR, chest compression depth was titrated to peak aortic compression pressure of 100 mm Hg, and vasopressor administration was titrated to coronary perfusion pressure greater than or equal to 20 mm Hg. Defibrillation attempts began after 10 minutes of resuscitation. The primary outcome was 45-minute survival. MEASUREMENTS AND MAIN RESULTS: The iNO group had higher rates of 45-minute survival (10 of 10 vs. 3 of 11; P = 0.001). During cardiopulmonary resuscitation, the iNO group had lower pulmonary artery relaxation pressure (mean ± SEM, 10.9 ± 2.4 vs. 18.4 ± 2.4 mm Hg; P = 0.03), higher coronary perfusion pressure (21.1 ± 1.5 vs. 16.9 ± 1.0 mm Hg; P = 0.005), and higher aortic relaxation pressure (36.6 ± 1.6 vs. 30.4 ± 1.1 mm Hg; P < 0.001) despite shallower chest compressions (5.88 ± 0.25 vs. 6.46 ± 0.40 cm; P = 0.02) and fewer vasopressor doses in the first 10 minutes (median, 4 [interquartile range, 3-4] vs. 5 [interquartile range, 5-6], P = 0.03). CONCLUSIONS: The addition of iNO to HD-CPR in LPS-induced shock-associated cardiac arrest improved short-term survival and intraarrest hemodynamics.
Assuntos
Reanimação Cardiopulmonar/métodos , Parada Cardíaca/etiologia , Parada Cardíaca/terapia , Óxido Nítrico/uso terapêutico , Choque/complicações , Vasodilatadores/uso terapêutico , Animais , Terapia Combinada/métodos , Modelos Animais de Doenças , Sequestradores de Radicais Livres/uso terapêutico , SuínosRESUMO
Peers are an important adjunct to the public mental health service system, and are being increasingly utilized across the country as a cost-effective solution to workforce shortages. Despite the tremendous growth of peer-delivered support over the past two decades, it has only been within the past few years that peer programs have been the subject of empirical inquiry. The purpose of this study was to examine the prevalence and characteristics of peer-delivered parenting programs across the New York State public mental health service system. We surveyed 46 family peer organizations across New York State regarding their delivery of structured peer-delivered parenting programs. Thirty-four (76%) completed the questionnaire, and of them, 18 (53%) delivered a parenting program. Subsequent interviews with seven of the 18 organizations revealed peer organizations had been delivering eight unique parenting programs for upwards of two decades. Additionally, organizations offered multiple supports to families to participate. Training, supervision, and issues around fidelity are discussed, as well as the implications of this study for states utilizing a peer workforce.
Assuntos
Educação não Profissionalizante/métodos , Grupo Associado , Adolescente , Criança , Pré-Escolar , Educação não Profissionalizante/estatística & dados numéricos , Humanos , Lactente , Recém-Nascido , Serviços de Saúde Mental , New York , Pais/educação , Pais/psicologia , Inquéritos e QuestionáriosRESUMO
The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB-TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB-TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD.
Assuntos
DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transposases/metabolismo , Sítios de Ligação Microbiológicos/fisiologia , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligação Proteica , Transposases/genéticaRESUMO
The transposon Tn7 transposase that recognizes the transposon ends and mediates breakage and joining is heteromeric. It contains the Tn7-encoded proteins TnsB, which binds specifically to the transposon ends and carries out breakage and joining at the 3' ends, and TnsA, which carries out breakage at the 5' ends of Tn7. TnsA apparently does not bind specifically to DNA, and we have hypothesized that it is recruited to the ends by interaction with TnsB. In this work, we show that TnsA and TnsB interact directly and identify several TnsA and TnsB amino acids involved in this interaction. We also show that TnsA can stimulate two key activities of TnsB, specific binding to the ends and pairing of the Tn7 ends. The ends of Tn7 are structurally asymmetric (i.e., contain different numbers of TnsB-binding sites), and Tn7 also is functionally asymmetric, inserting into its specific target site, attachment site attTn7 (attTn7) in a single orientation. Moreover, Tn7 elements containing two Tn7 right ends can transpose, but elements with two Tn7 left ends cannot. We show here that TnsA + TnsB are unable to pair the ends of a Tn7 element containing two Tn7 left ends. This pairing defect likely contributes to the inability of Tn7 elements with two Tn7 left ends to transpose.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Subunidades Proteicas/metabolismo , Transposases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Southern Blotting , Western Blotting , Primers do DNA/genética , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Subunidades Proteicas/genética , Transposases/genéticaRESUMO
A revelation of the genomic age has been the contributions of the mobile DNA segments called transposable elements to chromosome structure, function, and evolution in virtually all organisms. Substantial fractions of vertebrate genomes derive from transposable elements, being dominated by retroelements that move via RNA intermediates. Although many of these elements have been inactivated by mutation, several active retroelements remain. Vertebrate genomes also contain substantial quantities and a high diversity of cut-and-paste DNA transposons, but no active representative of this class has been identified in mammals. Here we show that a cut-and-paste element called piggyBat, which has recently invaded the genome of the little brown bat (Myotis lucifugus) and is a member of the piggyBac superfamily, is active in its native form in transposition assays in bat and human cultured cells, as well as in the yeast Saccharomyces cerevisiae. Our study suggests that some DNA transposons are still actively shaping some mammalian genomes and reveals an unprecedented opportunity to study the mechanism, regulation, and genomic impact of cut-and-paste transposition in a natural mammalian host.
Assuntos
Quirópteros/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Genoma/genética , Animais , Sequência de Bases , Células Cultivadas , Biologia Computacional , Primers do DNA/genética , Elementos de DNA Transponíveis/fisiologia , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Saccharomyces cerevisiaeRESUMO
Chromosome structure and function are influenced by transposable elements, which are mobile DNA segments that can move from place to place. hAT elements are a superfamily of DNA cut and paste elements that move by excision and integration. We have characterized two hAT elements, TcBuster and Space Invaders (SPIN), that are members of a recently described subfamily of hAT elements called Buster elements. We show that TcBuster, from the red flour beetle Tribolium castaneum, is highly active in human cells. SPIN elements are currently inactive elements that were recently highly active in multiple vertebrate genomes, and the high level of sequence similarity across widely diverged species and patchy phylogenetic distribution suggest that they may have moved between genomes by horizontal transfer. We have generated an intact version of this element, SPIN(ON), which is highly active in human cells. In vitro analysis of TcBuster and SPIN(ON) shows that no proteins other than transposase are essential for recombination, a property that may contribute to the ability of SPIN to successfully invade multiple organisms. We also analyze the target site preferences of de novo insertions in the human genome of TcBuster and SPIN(ON) and compare them with the preferences of Sleeping Beauty and piggyBac, showing that each superfamily has a distinctive pattern of insertion. The high-frequency transposition of both TcBuster and SPIN(ON) suggests that these transposon systems offer powerful tools for genome engineering. Finally, we describe a Saccharomyces cerevisiae assay for TcBuster that will provide a means for isolation of hyperactive and other interesting classes of transposase mutants.
Assuntos
Elementos de DNA Transponíveis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Transferência Genética Horizontal , Genes de Insetos , Engenharia Genética , Células HeLa , Humanos , Dados de Sequência Molecular , Filogenia , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transposases/metabolismo , Tribolium/genéticaRESUMO
The transposon piggyBac is being used increasingly for genetic studies. Here, we describe modified versions of piggyBac transposase that have potentially wide-ranging applications, such as reversible transgenesis and modified targeting of insertions. piggyBac is distinguished by its ability to excise precisely, restoring the donor site to its pretransposon state. This characteristic makes piggyBac useful for reversible transgenesis, a potentially valuable feature when generating induced pluripotent stem cells without permanent alterations to genomic sequence. To avoid further genome modification following piggyBac excision by reintegration, we generated an excision competent/integration defective (Exc(+)Int(-)) transposase. Our findings also suggest the position of a target DNA-transposase interaction. Another goal of genome engineering is to develop reagents that can guide transgenes to preferred genomic regions. Others have shown that piggyBac transposase can be active when fused to a heterologous DNA-binding domain. An Exc(+)Int(-) transposase, the intrinsic targeting of which is defective, might also be a useful intermediate in generating a transposase whose integration activity could be rescued and redirected by fusion to a site-specific DNA-binding domain. We show that fusion to two designed zinc finger proteins rescued the Int(-) phenotype. Successful guided transgene integration into genomic DNA would have broad applications to gene therapy and molecular genetics. Thus, an Exc(+)Int(-) transposase is a potentially useful reagent for genome engineering and provides insight into the mechanism of transposase-target DNA interaction.
Assuntos
Elementos de DNA Transponíveis/genética , Engenharia Genética/métodos , Proteínas do Tecido Nervoso/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Técnicas de Transferência de Genes , Genoma Humano/genética , Células HEK293 , Células HeLa , Humanos , Mamíferos , Dados de Sequência Molecular , Mutagênese Insercional/métodos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Dedos de Zinco/genéticaRESUMO
DNA transposons have been widely used for transgenesis and insertional mutagenesis in various organisms. Among the transposons active in mammalian cells, the moth-derived transposon piggyBac is most promising with its highly efficient transposition, large cargo capacity, and precise repair of the donor site. Here we report the generation of a hyperactive piggyBac transposase. The active transposition of piggyBac in multiple organisms allowed us to screen a transposase mutant library in yeast for hyperactive mutants and then to test candidates in mouse ES cells. We isolated 18 hyperactive mutants in yeast, among which five were also hyperactive in mammalian cells. By combining all mutations, a total of 7 aa substitutions, into a single reading frame, we generated a unique hyperactive piggyBac transposase with 17-fold and ninefold increases in excision and integration, respectively. We showed its applicability by demonstrating an increased efficiency of generation of transgene-free mouse induced pluripotent stem cells. We also analyzed whether this hyperactive piggyBac transposase affects the genomic integrity of the host cells. The frequency of footprints left by the hyperactive piggyBac transposase was as low as WT transposase (~1%) and we found no evidence that the expression of the transposase affects genomic integrity. This hyperactive piggyBac transposase expands the utility of the piggyBac transposon for applications in mammalian genetics and gene therapy.
Assuntos
Elementos de DNA Transponíveis/genética , Células-Tronco Embrionárias/metabolismo , Mutagênese Insercional , Transposases/metabolismo , Processamento Alternativo/genética , Animais , Células Cultivadas , Hibridização Genômica Comparativa , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Genoma/genética , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Genéticos , Mariposas/genética , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transposases/genéticaRESUMO
Quality indicators for programs integrating parent-delivered family support services for children's mental health have not been systematically developed. Increasing emphasis on accountability under the Affordable Care Act highlights the importance of quality-benchmarking efforts. Using a modified Delphi approach, quality indicators were developed for both program level and family support specialist level practices. These indicators were pilot tested with 21 community-based mental health programs. Psychometric properties of these indicators are reported; variations in program and family support specialist performance suggest the utility of these indicators as tools to guide policies and practices in organizations that integrate parent-delivered family support service components.
Assuntos
Serviços Comunitários de Saúde Mental/organização & administração , Terapia Familiar/organização & administração , Transtornos Mentais/terapia , Equipe de Assistência ao Paciente/organização & administração , Grupo Associado , Indicadores de Qualidade em Assistência à Saúde/organização & administração , Apoio Social , Adolescente , Benchmarking/organização & administração , Criança , Pré-Escolar , Comportamento Cooperativo , Técnica Delphi , Humanos , Comunicação Interdisciplinar , Projetos Piloto , Estados UnidosRESUMO
The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.
Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos , Genoma , Animais , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Loci Gênicos , Humanos , Hidrolases/genética , CamundongosRESUMO
Transposons are mobile genetic elements that are an important source of genetic variation and are useful tools for genome engineering, mutagenesis screens, and vectors for transgenesis including gene therapy. We have used second-generation sequencing to analyze ≈2 × 10(5) unique de novo transposon insertion sites of the transposon Hermes in the Saccharomyces cerevisiae genome from both in vitro transposition reactions by using purified yeast genomic DNA, to better characterize intrinsic sequence specificity, and sites recovered from in vivo transposition events, to characterize the effect of intracellular factors such as chromatin on target site selection. We find that Hermes transposon targeting in vivo is profoundly affected by chromatin structure: The subset of genome-wide target sites used in vivo is strongly associated (P < 2e-16 by Fisher's exact test) with nucleosome-free chromatin. Our characterization of the insertion site preferences of Hermes not only assists in the future use of this transposon as a molecular biology tool but also establishes methods to more fully determine targeting mechanisms of other transposons. We have also discovered a long-range sequence motif that defines S. cerevisiae nucleosome-free regions.
Assuntos
Elementos de DNA Transponíveis/genética , DNA Fúngico/genética , Genoma Fúngico/fisiologia , Nucleossomos/genética , Saccharomyces cerevisiae/genética , DNA Fúngico/metabolismo , Estudo de Associação Genômica Ampla , Nucleossomos/metabolismoRESUMO
DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.
Assuntos
Elementos de DNA Transponíveis/genética , DNA/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Catálise , Sequência Conservada , Quebras de DNA de Cadeia Dupla , Insetos , Dados de Sequência Molecular , Mutagênese Insercional , Mutação/genética , Conformação de Ácido Nucleico , Saccharomyces cerevisiae/genética , Transposases/metabolismoRESUMO
BACKGROUND: The piRNA pathway has been shown in model organisms to be involved in silencing of transposons thereby providing genome stability. In D. melanogaster the majority of piRNAs map to these sequences. The medically important mosquito species Aedes aegypti has a large genome size, a high transposon load which includes Miniature Inverted repeat Transposable Elements (MITES) and an expansion of the piRNA biogenesis genes. Studies of transgenic lines of Ae. aegypti have indicated that introduced transposons are poorly remobilized and we sought to explore the basis of this. We wished to analyze the piRNA profile of Ae. aegypti and thereby determine if it is responsible for transposon silencing in this mosquito. RESULTS: Estimated piRNA sequence diversity was comparable between Ae. aegypti and D. melanogaster, but surprisingly only 19% of mosquito piRNAs mapped to transposons compared to 51% for D. melanogaster. Ae. aegypti piRNA clusters made up a larger percentage of the total genome than those of D. melanogaster but did not contain significantly higher percentages of transposon derived sequences than other regions of the genome. Ae. aegypti contains a number of protein coding genes that may be sources of piRNA biogenesis with two, traffic jam and maelstrom, implicated in this process in model organisms. Several genes of viral origin were also targeted by piRNAs. Examination of six mosquito libraries that had previously been transformed with transposon derived sequence revealed that new piRNA sequences had been generated to the transformed sequences, suggesting that they may have stimulated a transposon inactivation mechanism. CONCLUSIONS: Ae. aegypti has a large piRNA complement that maps to transposons but primarily gene sequences, including many viral-derived sequences. This, together the more uniform distribution of piRNA clusters throughout its genome, suggest that some aspects of the piRNA system differ between Ae. aegypti and D. melanogaster.
Assuntos
Aedes/genética , Elementos de DNA Transponíveis , Genoma , RNA Interferente Pequeno/genética , Animais , Inativação GênicaRESUMO
Mobile elements and their inactive remnants account for large proportions of most eukaryotic genomes, where they have had central roles in genome evolution. Over 50 years ago, McClintock reported a form of stress-induced genome instability in maize in which discrete DNA segments move between chromosomal locations. Our current mechanistic understanding of enzymes catalyzing transposition is largely limited to prokaryotic transposases. The Hermes transposon from the housefly is part of the eukaryotic hAT superfamily that includes hobo from Drosophila, McClintock's maize Activator and Tam3 from snapdragon. We report here the three-dimensional structure of a functionally active form of the transposase from Hermes at 2.1-A resolution. The Hermes protein has some structural features of prokaryotic transposases, including a domain with a retroviral integrase fold. However, this domain is disrupted by the insertion of an additional domain. Finally, transposition is observed only when Hermes assembles into a hexamer.