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1.
Yi Chuan ; 41(5): 422-429, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31106778

RESUMO

The type2 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.


Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Edição de Genes , Genoma de Inseto , Transposases/genética , Animais
2.
Nat Commun ; 10(1): 1930, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036827

RESUMO

Many chromatin features play critical roles in regulating gene expression. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells.


Assuntos
Cromatina/química , Epigenômica/métodos , Perfilação da Expressão Gênica/métodos , Análise de Célula Única/métodos , Coloração e Rotulagem/métodos , Cromatina/metabolismo , Regulação da Expressão Gênica , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Código das Histonas , Histonas/genética , Histonas/metabolismo , Humanos , Células K562 , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transposases/genética , Transposases/metabolismo
3.
Methods Cell Biol ; 151: 219-235, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948010

RESUMO

Programs of gene transcription are controlled by cis-acting DNA elements, including enhancers, silencers, and promoters. Local accessibility of chromatin has proven to be a highly informative structural feature for identifying such regulatory elements, which tend to be relatively open due to their interactions with proteins. Recently, ATAC-seq (assay for transposase-accessible chromatin using sequencing) has emerged as one of the most powerful approaches for genome-wide chromatin accessibility profiling. This method assesses DNA accessibility using hyperactive Tn5 transposase, which simultaneously cuts DNA and inserts sequencing adaptors, preferentially in regions of open chromatin. ATAC-seq is a relatively simple procedure which can be applied to only a few thousand cells. It is well-suited to developing embryos of sea urchins and other echinoderms, which are a prominent experimental model for understanding the genomic control of animal development. In this chapter, we present a protocol for applying ATAC-seq to embryonic cells of sea urchins.


Assuntos
Cromatina/genética , Equinodermos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Animais , Equinodermos/crescimento & desenvolvimento , Elementos Facilitadores Genéticos/genética , Regiões Promotoras Genéticas/genética , Sequências Reguladoras de Ácido Nucleico/genética , Elementos Silenciadores Transcricionais/genética , Transposases/química , Transposases/genética
4.
PLoS Genet ; 15(1): e1007883, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30615607

RESUMO

Intercellular communication and self-recognition are critical for coordinating cooperative and competitive behaviors during sociomicrobiological community development. Contact-dependent growth inhibition (CDI) proteins are polymorphic toxin delivery systems that inhibit the growth of non-self neighboring bacteria that lack the appropriate immunity protein. In Burkholderia thailandensis, CDI system proteins (encoded by bcpAIOB genes) also induce cooperative behaviors among sibling (self) cells, a phenomenon called contact-dependent signaling (CDS). Here we describe a mobile genetic element (MGE) that carries the bcpAIOB genes in B. thailandensis E264. It is a ~210 kb composite transposon with insertion sequence (IS) elements at each end. Although the ISs are most similar to IS2 of Escherichia coli, the transposase-dependent intermediate molecule displays characteristics more similar to those of the IS26 translocatable unit (TU). A reaction requiring only the "left" IS-encoded transposase results in formation of an extrachromosomal circular dsDNA intermediate ("the megacircle") composed of the left IS and the sequences intervening between the ISs. Insertion of the megacircle into the chromosome occurs next to a pre-existing copy of an IS2-like element, recreating a functional composite transposon. We found that BcpA activity is required for megacircle formation, and in turn, megacircle formation is required for CDS phenotypes. Our data support a model in which the bcpAIOB genes function as both helping and harming greenbeard genes, simultaneously enhancing the fitness of self bacteria that possess the same allele plus tightly linked genes that mediate cooperative behaviors, and killing non-self bacteria that do not possess the same bcpAIOB allele. Mobility of the megacircle between cells could allow bacteria invading a community to be converted to self, and would facilitate propagation of the bcpAIOB genes in the event that the invading strain is capable of overtaking the resident community.


Assuntos
Proteínas de Bactérias/genética , Burkholderia/genética , Elementos de DNA Transponíveis/genética , Sequências Repetitivas Dispersas/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Inibição de Contato/genética , DNA Circular/genética , Plasmídeos/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Transdução de Sinais , Transposases/genética
5.
BMC Genomics ; 20(1): 25, 2019 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626325

RESUMO

BACKGROUND: Orchids produce a colorless protocorm by symbiosis with fungi upon seed germination. For mass production of orchids, the prevailing approaches are both generation of protocorm-like bodies (PLBs) from callus and multiplication of adventitious buds on inflorescence. However, somaclonal variations occur during micropropagation. RESULTS: We isolated the two most expressed transposable elements belonging to P Instability Factor (PIF)-like transposons. Among them, a potential autonomous element was identified by similarity analysis against the whole-genome sequence of Phalaenopsis equestris and named PePIF1. It contains a 19-bp terminal inverted repeat flanked by a 3-bp target site duplication and two coding regions encoding ORF1- and transposase-like proteins. Phylogenetic analysis revealed that PePIF1 belongs to a new P-lineage of PIF. Furthermore, two distinct families, PePIF1a and PePIF1b, with 29 and 37 putative autonomous elements, respectively, were isolated, along with more than 3000 non-autonomous and miniature inverted-repeat transposable element (MITE)-like elements. Among them, 828 PePIF1-related elements were inserted in 771 predicted genes. Intriguingly, PePIF1 was transposed in the somaclonal variants of Phalaenopsis cultivars, as revealed by transposon display, and the newly inserted genes were identified and sequenced. CONCLUSION: A PIF-like element, PePIF1, was identified in the Phalaenopsis genome and actively transposed during micropropagation. With the identification of PePIF1, we have more understanding of the Phalaenopsis genome structure and somaclonal variations during micropropagation for use in orchid breeding and production.


Assuntos
Elementos de DNA Transponíveis/genética , Orchidaceae/genética , Filogenia , Genoma de Planta/genética , Mutagênese Insercional/genética , Fases de Leitura Aberta , Sequências Repetidas Terminais/genética , Transposases/genética
7.
Gene ; 679: 65-72, 2018 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-30171941

RESUMO

Transposable elements (TEs) are mobile DNA sequences on genomes. Some elements are able to transpose in somatic cells, a process known as somatic transposition (ST), which has been associated with detrimental biological effects. The mariner-Mos1 element of Drosophila promotes transposition in somatic and germline cells and is an excellent model for studies related to the biological consequence of somatic excision (SE). In this work, we used temperature stress to induce increasing transposition of mariner-Mos1 during different stages of the development of D. simulans, aiming to quantify SE during lifespan. Furthermore, strains of D. melanogaster exhibiting differential expression of mariner-Mos1 were employed for estimating some biological consequences of mariner mobilization. It is shown that SE of mariner-Mos1 was not constant during development; the larval phase had the highest rates while the pupal stage exhibited lower rates, and in the embryonic stage, no difference was detected. SE can be detrimental, as suggested by correlation in SE level and reduction in behavioral activities and embryonic viability. This study showed that mariner-Mos1 SE accumulates during the Drosophila life cycle, and can be involved in detrimental effects.


Assuntos
Elementos de DNA Transponíveis , Drosophila melanogaster/crescimento & desenvolvimento , Estresse Fisiológico , Animais , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Fenótipo , Temperatura Ambiente , Transposases/genética
8.
Genes Genomics ; 40(5): 485-495, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29892960

RESUMO

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated "cut and paste" mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.


Assuntos
Bombyx/genética , Proteínas de Ligação a DNA/genética , Transposases/genética , Animais , Elementos de DNA Transponíveis/genética , Genoma de Inseto/genética , Estudo de Associação Genômica Ampla/métodos , Filogenia
9.
PLoS One ; 13(5): e0197012, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29723287

RESUMO

The rickettsiae are obligate intracellular alphaproteobacteria that exhibit a complex infectious life cycle in both arthropod and mammalian hosts. As obligate intracellular bacteria, rickettsiae are highly adapted to living inside a variety of host cells, including vascular endothelial cells during mammalian infection. Although it is assumed that the rickettsiae produce numerous virulence factors that usurp or disrupt various host cell pathways, they have been challenging to genetically manipulate to identify the key bacterial factors that contribute to infection. Motivated to overcome this challenge, we sought to expand the repertoire of available rickettsial loss-of-function mutants, using an improved mariner-based transposon mutagenesis scheme. Here, we present the isolation of over 100 transposon mutants in the spotted fever group species Rickettsia parkeri. Transposon insertions disrupted genes whose products are implicated in a variety of pathways, including bacterial replication and metabolism, the type IV secretion system, factors with previously established roles in host cell interactions and pathogenesis, or are of unknown function. Given the need to identify critical virulence factors, forward genetic screens such as this will provide an excellent platform to more directly investigate rickettsial biology and pathogenesis.


Assuntos
Proteínas de Bactérias/genética , Elementos de DNA Transponíveis , Rickettsia/genética , Sistemas de Secreção Tipo IV/genética , Fatores de Virulência/genética , Animais , Proteínas de Bactérias/metabolismo , Cercopithecus aethiops , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutagênese , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Rickettsia/metabolismo , Rickettsia/patogenicidade , Transposases/genética , Transposases/metabolismo , Sistemas de Secreção Tipo IV/metabolismo , Células Vero , Fatores de Virulência/metabolismo
10.
Biochemistry ; 57(20): 2913-2922, 2018 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-29750515

RESUMO

The movement of the piggyBac transposon is mediated through its cognate transposase. The piggyBac transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac transposase. Surprisingly, the monomeric constructs exhibit >2-fold enhancement in the excision activity of the enzyme. Overall, our studies suggest that dimerization attenuates the excision activity of the piggyBac transposase. This attribute of the piggyBac transposase may serve to prevent excessive transposition of the piggyBac transposon that might be catastrophic for the host cell.


Assuntos
Elementos de DNA Transponíveis/genética , Domínios RING Finger/genética , Transposases/química , Dimerização , Vetores Genéticos/química , Vetores Genéticos/genética , Mutagênese Insercional , Transposases/genética
11.
Tissue Cell ; 51: 49-55, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29622087

RESUMO

The objective of this study was to optimise the electroporation conditions for efficient integration of Venus construct in buffalo fetal fibroblasts using Sleeping Beauty (SB) based transposition and to produce Venus expressing transgenic cloned embryos through handmade cloning (HMC) approach. Primary culture of buffalo fetal fibroblast cells was established and subsequently cultured cells were co-transfected with Venus and helper plasmid at different combinations of electroporation condition. In different combinations of voltage, time and plasmid dose, we observed that 300 V, single pulse for 10 ms in 2 mm cuvette and 1.5-2.0 µg transposons with 200-300 ng transposase dose was optimum for expressing Venus fluorescence in cells via electroporation. After electroporation, the cells were cultured for 2-3 days and then Venus expressing cells were picked with the help of a Pasteur pipette under the fluorescence microscope to enrich them through single cell culture method before using as donor cells for HMC. In vitro matured oocytes were reconstructed with either transfected or non-transfected buffalo somatic cells by electric fusion followed by activation. The reconstructed, activated embryos were cultured in 400 µL of Research Vitro Cleave medium supplemented with 1% fatty acid-free BSA in 4-well dish, covered with mineral oil and incubated in an incubator (5% CO2 in air) at 38.5 °C for 8 days and the developmental competence was observed. The percentage of cleaved, 4-8 and 8-16 cells stage embryos generated through Venus expressing cells were comparable with control, whereas, the morula (21.0 vs 53.0%) and blastocysts (10.5 vs 30.6%) produced through Venus expressing cells was found low as compared to control. These results indicate that fetal fibroblasts transfected with Venus could be used as donor cells for buffalo cloning and that Venus gene can be safely used as a marker of foreign gene in buffalo transgenesis.


Assuntos
Animais Geneticamente Modificados/genética , Elementos de DNA Transponíveis/genética , Engenharia Genética/métodos , Transposases/genética , Animais , Búfalos , Células Cultivadas , Clonagem de Organismos/métodos , Eletroporação/métodos , Embrião de Mamíferos , Fibroblastos , Corantes Fluorescentes , Técnicas de Transferência Nuclear
12.
BMC Genomics ; 19(1): 169, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29490630

RESUMO

BACKGROUND: ATAC-seq (Assays for Transposase-Accessible Chromatin using sequencing) is a recently developed technique for genome-wide analysis of chromatin accessibility. Compared to earlier methods for assaying chromatin accessibility, ATAC-seq is faster and easier to perform, does not require cross-linking, has higher signal to noise ratio, and can be performed on small cell numbers. However, to ensure a successful ATAC-seq experiment, step-by-step quality assurance processes, including both wet lab quality control and in silico quality assessment, are essential. While several tools have been developed or adopted for assessing read quality, identifying nucleosome occupancy and accessible regions from ATAC-seq data, none of the tools provide a comprehensive set of functionalities for preprocessing and quality assessment of aligned ATAC-seq datasets. RESULTS: We have developed a Bioconductor package, ATACseqQC, for easily generating various diagnostic plots to help researchers quickly assess the quality of their ATAC-seq data. In addition, this package contains functions to preprocess aligned ATAC-seq data for subsequent peak calling. Here we demonstrate the utilities of our package using 25 publicly available ATAC-seq datasets from four studies. We also provide guidelines on what the diagnostic plots should look like for an ideal ATAC-seq dataset. CONCLUSIONS: This software package has been used successfully for preprocessing and assessing several in-house and public ATAC-seq datasets. Diagnostic plots generated by this package will facilitate the quality assessment of ATAC-seq data, and help researchers to evaluate their own ATAC-seq experiments as well as select high-quality ATAC-seq datasets from public repositories such as GEO to avoid generating hypotheses or drawing conclusions from low-quality ATAC-seq experiments. The software, source code, and documentation are freely available as a Bioconductor package at https://bioconductor.org/packages/release/bioc/html/ATACseqQC.html .


Assuntos
Biologia Computacional/métodos , Análise de Sequência de DNA/métodos , Software , Sítios de Ligação , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala , Mutagênese Insercional , Sítio de Iniciação de Transcrição , Transposases/genética , Transposases/metabolismo , Navegador
13.
Braz. j. microbiol ; 49(1): 138-143, Jan.-Mar. 2018. tab, graf
Artigo em Inglês | LILACS | ID: biblio-889188

RESUMO

ABSTRACT Q fever is a worldwide zoonosis caused by Coxiella burnetii—a small obligate intracellular Gram-negative bacterium found in a variety of animals. It is transmitted to humans by inhalation of contaminated aerosols from urine, feces, milk, amniotic fluid, placenta, abortion products, wool, and rarely by ingestion of raw milk from infected animals. Nested PCR can improve the sensitivity and specificity of testing while offering a suitable amplicon size for sequencing. Serial dilutions were performed tenfold to test the limit of detection, and the result was 10× detection of C. burnetti DNA with internal nested PCR primers relative to trans-PCR. Different biological samples were tested and identified only in nested PCR. This demonstrates the efficiency and effectiveness of the primers. Of the 19 samples, which amplify the partial sequence of C. burnetii, 12 were positive by conventional PCR and nested PCR. Seven samples—five spleen tissue samples from rodents and two tick samples—were only positive in nested PCR. With these new internal primers for trans-PCR, we demonstrate that our nested PCR assay for C. burnetii can achieve better results than conventional PCR.


Assuntos
Humanos , Proteínas de Bactérias/genética , Coxiella burnetii/isolamento & purificação , Elementos de DNA Transponíveis , Febre/microbiologia , Reação em Cadeia da Polimerase/métodos , Transposases/genética , Proteínas de Bactérias/metabolismo , Coxiella burnetii/classificação , Coxiella burnetii/genética , Transposases/metabolismo
14.
J Vis Exp ; (131)2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29364270

RESUMO

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a "cut and paste" SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.


Assuntos
Elementos de DNA Transponíveis , Vetores Genéticos/genética , Lentivirus/genética , Transposases/genética , Animais , Células HEK293 , Células HeLa , Humanos , Transfecção
15.
Genetica ; 146(2): 243-247, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29352755

RESUMO

The integrase and transposase enzymes of retrovirus and transposons, respectively, share the catalytic DDE domain. In vitro assays showed that inhibitors of HIV-1 integrase generally inhibit the mariner Mos1 transposase. Using a Drosophila strain in which the mobilisation of the mariner element can be quantified by mosaic eyes, we showed that flies maintained in medium containing 210 µM to 4 mM of raltegravir, or 1 or 2 mM of dolutegravir, which are HIV-1 integrase inhibitor used in AIDS treatment, have 23-33% less somatic mobilisation in mosaic eyes when treated with raltegravir and 28-32% when treated with dolutegravir. The gene expression of the mariner transposase gene, estimated by qPCR, is similar among treated and control flies. The results suggest that in vivo assays using Drosophila can be used as a primary screening of inhibitory drugs for transposase and retroviral integrase. The advantages of this assay are that it is easy, quick, cheap and is an in vivo test, meaning that the tested substance has to have been taken in by cells and has arrived at the target site, which is not the case when in vitro assays are applied.


Assuntos
Proteínas de Ligação a DNA/genética , Inibidores de Integrase de HIV/farmacologia , HIV-1 , Transposases/genética , Animais , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/metabolismo , Drosophila/anatomia & histologia , Drosophila/genética , Drosophila/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Compostos Heterocíclicos com 3 Anéis/farmacologia , Fenótipo , Raltegravir Potássico/farmacologia , Transposases/metabolismo
16.
J Cell Physiol ; 233(2): 990-1004, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28387938

RESUMO

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species.


Assuntos
Reprogramação Celular , Elementos de DNA Transponíveis , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Transcrição Genética , Ativação Transcricional , Animais , Diferenciação Celular , Células Cultivadas , Técnicas de Reprogramação Celular , Galinhas , Técnicas de Cocultura , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Fenótipo , Análise de Sequência de DNA , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma , Transfecção , Transposases/genética , Transposases/metabolismo
17.
G3 (Bethesda) ; 8(1): 79-89, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29118030

RESUMO

Efficient preparation of high-quality sequencing libraries that well represent the biological sample is a key step for using next-generation sequencing in research. Tn5 enables fast, robust, and highly efficient processing of limited input material while scaling to the parallel processing of hundreds of samples. Here, we present a robust Tn5 transposase purification strategy based on an N-terminal His6-Sumo3 tag. We demonstrate that libraries prepared with our in-house Tn5 are of the same quality as those processed with a commercially available kit (Nextera XT), while they dramatically reduce the cost of large-scale experiments. We introduce improved purification strategies for two versions of the Tn5 enzyme. The first version carries the previously reported point mutations E54K and L372P, and stably produces libraries of constant fragment size distribution, even if the Tn5-to-input molecule ratio varies. The second Tn5 construct carries an additional point mutation (R27S) in the DNA-binding domain. This construct allows for adjustment of the fragment size distribution based on enzyme concentration during tagmentation, a feature that opens new opportunities for use of Tn5 in customized experimental designs. We demonstrate the versatility of our Tn5 enzymes in different experimental settings, including a novel single-cell polyadenylation site mapping protocol as well as ultralow input DNA sequencing.


Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação Puntual , Proteínas Recombinantes de Fusão/genética , Transposases/genética , Sequência de Bases , Clonagem Molecular/métodos , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Poliadenilação , Ligação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transposases/metabolismo
18.
Braz J Microbiol ; 49(1): 138-143, 2018 Jan - Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28899604

RESUMO

Q fever is a worldwide zoonosis caused by Coxiella burnetii-a small obligate intracellular Gram-negative bacterium found in a variety of animals. It is transmitted to humans by inhalation of contaminated aerosols from urine, feces, milk, amniotic fluid, placenta, abortion products, wool, and rarely by ingestion of raw milk from infected animals. Nested PCR can improve the sensitivity and specificity of testing while offering a suitable amplicon size for sequencing. Serial dilutions were performed tenfold to test the limit of detection, and the result was 10× detection of C. burnetti DNA with internal nested PCR primers relative to trans-PCR. Different biological samples were tested and identified only in nested PCR. This demonstrates the efficiency and effectiveness of the primers. Of the 19 samples, which amplify the partial sequence of C. burnetii, 12 were positive by conventional PCR and nested PCR. Seven samples-five spleen tissue samples from rodents and two tick samples-were only positive in nested PCR. With these new internal primers for trans-PCR, we demonstrate that our nested PCR assay for C. burnetii can achieve better results than conventional PCR.


Assuntos
Proteínas de Bactérias/genética , Coxiella burnetii/isolamento & purificação , Elementos de DNA Transponíveis , Febre/microbiologia , Reação em Cadeia da Polimerase/métodos , Transposases/genética , Proteínas de Bactérias/metabolismo , Coxiella burnetii/classificação , Coxiella burnetii/genética , Humanos , Transposases/metabolismo
19.
RNA Biol ; 15(2): 176-181, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29120256

RESUMO

Bacterial transposons were long thought of as selfish mobile genetic elements that propagate at the expense of 'host' bacterium fitness. However, limited transposition can benefit the host organism by promoting DNA rearrangements and facilitating horizontal gene transfer. Here we discuss and provide context for our recently published work which reported the surprising finding that an otherwise dormant transposon, IS200, encodes a regulatory RNA in Salmonella Typhimurium. This previous work identified a trans-acting sRNA that is encoded in the 5'UTR of IS200 transposase mRNA (tnpA). This sRNA represses expression of genes encoded within Salmonella Pathogenicity Island 1 (SPI-1), and accordingly limits invasion into non-phagocytic cells in vitro. We present new data here that shows IS200 elements are important for colonization of the mouse gastrointestinal tract. We discuss our previous and current findings in the context of transposon biology and suggest that otherwise 'silent' transposons may in fact play an important role in controlling host gene expression.


Assuntos
Elementos de DNA Transponíveis , Pequeno RNA não Traduzido/genética , Salmonelose Animal/microbiologia , Salmonella typhimurium/patogenicidade , Transposases/genética , Regiões 5' não Traduzidas , Animais , Proteínas de Bactérias/genética , Regulação para Baixo , Trato Gastrointestinal/microbiologia , Regulação Bacteriana da Expressão Gênica , Camundongos , Salmonelose Animal/genética , Salmonella typhimurium/genética , Virulência
20.
Proc Natl Acad Sci U S A ; 114(49): E10550-E10559, 2017 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-29158416

RESUMO

To understand the success strategies of transposable elements (TEs) that attain high copy numbers, we analyzed two pairs of rice (Oryza sativa) strains, EG4/HEG4 and A119/A123, undergoing decades of rapid amplification (bursts) of the class 2 autonomous Ping element and the nonautonomous miniature inverted repeat transposable element (MITE) mPing Comparative analyses of whole-genome sequences of the two strain pairs validated that each pair has been maintained for decades as inbreds since divergence from their respective last common ancestor. Strains EG4 and HEG4 differ by fewer than 160 SNPs and a total of 264 new mPing insertions. Similarly, strains A119 and A123 exhibited about half as many SNPs (277) as new mPing insertions (518). Examination of all other potentially active TEs in these genomes revealed only a single new insertion out of ∼40,000 loci surveyed. The virtual absence of any new TE insertions in these strains outside the mPing bursts demonstrates that the Ping/mPing family gradually attains high copy numbers by maintaining activity and evading host detection for dozens of generations. Evasion is possible because host recognition of mPing sequences appears to have no impact on initiation or maintenance of the burst. Ping is actively transcribed, and both Ping and mPing can transpose despite methylation of terminal sequences. This finding suggests that an important feature of MITE success is that host recognition does not lead to the silencing of the source of transposase.


Assuntos
Elementos de DNA Transponíveis , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Oryza/genética , Transposases/genética , Variações do Número de Cópias de DNA , Metilação de DNA , Loci Gênicos , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/genética , Histonas/metabolismo , Mutagênese Insercional , Oryza/metabolismo , Melhoramento Vegetal , Polimorfismo de Nucleotídeo Único , Transposases/metabolismo
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