RESUMO
N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.
Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Desoxiadenosinas/genética , Genoma Mitocondrial , Proteínas Mitocondriais/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Sequência de Bases , Mapeamento Cromossômico , DNA/metabolismo , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.
Assuntos
Epigênese Genética/ética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Reprogramação Celular/genética , Impressões Digitais de DNA , Metilação de DNA/genética , Fibroblastos , Marcadores Genéticos , Humanos , Neoplasias Pulmonares/genética , Procedimentos Analíticos em Microchip/métodosRESUMO
BACKGROUND: Viral populations are complex, dynamic, and fast evolving. The evolution of groups of closely related viruses in a competitive environment is termed quasispecies. To fully understand the role that quasispecies play in viral evolution, characterizing the trajectories of viral genotypes in an evolving population is the key. In particular, long-range haplotype information for thousands of individual viruses is critical; yet generating this information is non-trivial. Popular deep sequencing methods generate relatively short reads that do not preserve linkage information, while third generation sequencing methods have higher error rates that make detection of low frequency mutations a bioinformatics challenge. Here we applied BAsE-Seq, an Illumina-based single-virion sequencing technology, to eight samples from four chronic hepatitis B (CHB) patients - once before antiviral treatment and once after viral rebound due to resistance. RESULTS: With single-virion sequencing, we obtained 248-8796 single-virion sequences per sample, which allowed us to find evidence for both hard and soft selective sweeps. We were able to reconstruct population demographic history that was independently verified by clinically collected data. We further verified four of the samples independently through PacBio SMRT and Illumina Pooled deep sequencing. CONCLUSIONS: Overall, we showed that single-virion sequencing yields insight into viral evolution and population dynamics in an efficient and high throughput manner. We believe that single-virion sequencing is widely applicable to the study of viral evolution in the context of drug resistance and host adaptation, allows differentiation between soft or hard selective sweeps, and may be useful in the reconstruction of intra-host viral population demographic history.
Assuntos
Evolução Molecular , Genoma Viral , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Hepatite B/virologia , Lamivudina/farmacologia , Vírion/genética , Alelos , Substituição de Aminoácidos , Biologia Computacional/métodos , Código de Barras de DNA Taxonômico , Farmacorresistência Viral/efeitos dos fármacos , Frequência do Gene , Hepatite B/tratamento farmacológico , Vírus da Hepatite B/isolamento & purificação , Humanos , Lamivudina/uso terapêutico , MutaçãoRESUMO
Insertions of the human-specific subfamily of LINE-1 (L1) retrotransposon are highly polymorphic across individuals and can critically influence the human transcriptome. We hypothesized that L1 insertions could represent genetic variants determining important human phenotypic traits, and performed an integrated analysis of L1 elements and single nucleotide polymorphisms (SNPs) in several human populations. We found that a large fraction of L1s were in high linkage disequilibrium with their surrounding genomic regions and that they were well tagged by SNPs. However, L1 variants were only partially captured by SNPs on standard SNP arrays, so that their potential phenotypic impact would be frequently missed by SNP array-based genome-wide association studies. We next identified potential phenotypic effects of L1s by looking for signatures of natural selection linked to L1 insertions; significant extended haplotype homozygosity was detected around several L1 insertions. This finding suggests that some of these L1 insertions may have been the target of recent positive selection.
Assuntos
Genoma Humano/genética , Desequilíbrio de Ligação/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Evolução Molecular , Regulação da Expressão Gênica/genética , Genética Populacional/métodos , Estudo de Associação Genômica Ampla , Haplótipos , Homozigoto , Humanos , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Seleção Genética/genéticaRESUMO
BACKGROUND: Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed. RESULTS: We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate. CONCLUSIONS: This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.
Assuntos
Elementos de DNA Transponíveis/genética , Polimorfismo Genético/genética , Alelos , Genótipo , Técnicas de Genotipagem/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodosRESUMO
Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.
Assuntos
Proteínas Cromossômicas não Histona/análise , Ensaios de Triagem em Larga Escala/economia , Histonas/análise , Técnicas Analíticas Microfluídicas/instrumentação , Peptídeos/análise , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Polarização de Fluorescência , Histonas/química , Histonas/metabolismo , Humanos , Ligantes , Técnicas Analíticas Microfluídicas/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Fatores de TempoRESUMO
Bacteria must accurately replicate and segregate their genetic information to ensure the production of viable daughter cells. The high fidelity of chromosome partitioning is achieved through mechanisms that coordinate cell division with DNA replication. We report that YycJ (WalJ), a predicted member of the metallo-ß-lactamase superfamily found in most low-G+C Gram-positive bacteria, contributes to the fidelity of cell division in Bacillus subtilis. B. subtilis ΔwalJ (ΔwalJ(Bsu)) mutants divide over unsegregated chromosomes more frequently than wild-type cells, and this phenotype is exacerbated when DNA replication is inhibited. Two lines of evidence suggest that WalJ(Bsu) and its ortholog in the Gram-positive pathogen Streptococcus pneumoniae, WalJ(Spn) (VicX), play a role in cell wall metabolism: (i) strains of B. subtilis and S. pneumoniae lacking walJ exhibit increased sensitivity to a narrow spectrum of cephalosporin antibiotics, and (ii) reducing the expression of a two-component system that regulates genes involved in cell wall metabolism, WalRK (YycFG), renders walJ essential for growth in B. subtilis, as observed previously with S. pneumoniae. Together, these results suggest that the enzymatic activity of WalJ directly or indirectly affects cell wall metabolism and is required for accurate coordination of cell division with DNA replication.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Divisão Celular , Parede Celular/metabolismo , Replicação do DNA , beta-Lactamases/metabolismo , Bacillus subtilis/citologia , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/enzimologia , Parede Celular/genética , beta-Lactamases/genéticaRESUMO
Cell viability depends on the stable transmission of genetic information to each successive generation. Therefore, in the event of intrinsic or extrinsic DNA damage, it is important that cell division be delayed until DNA repair has been completed. In Bacillus subtilis, this is accomplished in part by YneA, an inhibitor of division that is induced as part of the SOS response. We sought to gain insight into the mechanism by which YneA blocks cell division and the processes involved in shutting off YneA activity. Our data suggest that YneA is able to inhibit daughter cell separation as well as septum formation. YneA contains a LysM peptidoglycan binding domain and is predicted to be exported. We established that the YneA signal peptide is rapidly cleaved, resulting in secretion of YneA into the medium. Mutations within YneA affect both the rate of signal sequence cleavage and the activity of YneA. YneA does not stably associate with the cell wall and is rapidly degraded by extracellular proteases. Based on these results, we hypothesize that exported YneA is active prior to signal peptide cleavage and that proteolysis contributes to the inactivation of YneA. Finally, we identified mutations in the transmembrane segment of YneA that abolish the ability of YneA to inhibit cell division, while having little or no effect on YneA export or stability. These data suggest that protein-protein interactions mediated by the transmembrane region may be required for YneA activity.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Son Of Sevenless/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Divisão Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Genótipo , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Son Of Sevenless/genéticaRESUMO
The SOS response to DNA damage in bacteria is a well-known component of the complex transcriptional responses to genotoxic environmental stresses such as exposure to reactive oxygen species, alkylating agents, and many of the antibiotics targeting DNA replication. However, bacteria such as Bacillus subtilis also respond to conditions that perturb DNA replication via a transcriptional response mediated by the replication initiation protein DnaA. In addition to regulating the initiation of DNA replication, DnaA directly regulates the transcription of specific genes. Conditions that perturb DNA replication can trigger the accumulation of active DnaA, activating or repressing the transcription of genes in the DnaA regulon. We report here that simply growing B. subtilis in LB medium altered DnaA-dependent gene expression in a manner consistent with the accumulation of active DnaA and that this was part of a general transcriptional response to manganese limitation. The SOS response to DNA damage was not induced under these conditions. One of the genes positively regulated by DnaA in Bacillus subtilis encodes a protein that inhibits the initiation of sporulation, Sda. Sda expression was induced as cells entered stationary phase in LB medium but not in LB medium supplemented with manganese, and the induction of Sda inhibited sporulation-specific gene expression and the onset of spore morphogenesis. In the absence of Sda, manganese-limited cells initiated spore development but failed to form mature spores. These data highlight that DnaA-dependent gene expression may influence the response of bacteria to a range of environmental conditions, including conditions that are not obviously associated with genotoxic stress.
Assuntos
Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Manganês/farmacologia , Transcrição Gênica/efeitos dos fármacos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
In several bacterial species, the faithful completion of chromosome partitioning is known to be promoted by a conserved family of DNA translocases that includes Escherichia coli FtsK and Bacillus subtilis SpoIIIE. FtsK localizes at nascent division sites during every cell cycle and stimulates chromosome decatenation and the resolution of chromosome dimers formed by recA-dependent homologous recombination. In contrast, SpoIIIE localizes at sites where cells have divided and trapped chromosomal DNA in the membrane, which happens during spore development and under some conditions when DNA replication is perturbed. SpoIIIE completes chromosome segregation post-septationally by translocating trapped DNA across the membrane. Unlike E. coli, B. subtilis contains a second uncharacterized FtsK/SpoIIIE-like protein, SftA (formerly YtpS). We report that SftA plays a role similar to FtsK during each cell cycle but cannot substitute for SpoIIIE in rescuing trapped chromosomes. SftA colocalizes with FtsZ at nascent division sites but not with SpoIIIE at sites of chromosome trapping. SftA mutants divide over unsegregated chromosomes more frequently than wild-type unless recA is inactivated, suggesting that SftA, like FtsK, stimulates chromosome dimer resolution. Having two FtsK/SpoIIIE paralogues is not conserved among endospore-forming bacteria, but is highly conserved within several groups of soil- and plant-associated bacteria.
Assuntos
Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos BiológicosRESUMO
Histidine kinases are widely used by bacteria, fungi and plants to sense and respond to changing environmental conditions. Signals in addition to those directly sensed by the kinase are often integrated by proteins that fine-tune the biological response by modulating the activity of the kinase or its targets. The Bacillus subtilis histidine kinase KinA promotes the initiation of sporulation when nutrients are limiting, but sporulation can be delayed by two inhibitors of KinA, Sda (when DNA replication is perturbed) or KipI (under unknown conditions). We have identified residues in the dimerization/histidine-phosphotransfer (DHp) domain of KinA that are functionally important for inhibition by Sda and KipI and overlapping surface-exposed residues that lie close to or comprise the Sda binding site. Sda inhibits the intermolecular transfer of phosphate from the catalytic ATP-binding (CA) domain of KinA to the autophosphorylation site in the DHp domain when the domains are split into separate polypeptides, either by steric hindrance or by altering the conformation of the DHp domain. Sda also slows the rate of phosphotransfer from KinA approximately P to its target, Spo0F, consistent with our finding that a KinA residue important for Sda function overlaps with the predicted Spo0F binding site on KinA.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Histidina Quinase , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
The inaugural workshop "Deep Sequencing in Infectious Diseases: Immune and Pathogen Repertoires for the Improvement of Patient Outcomes" was held in Singapore on 13-14 October 2016. The aim of the workshop was to discuss the latest trends in using high-throughput sequencing, bioinformatics, and allied technologies to analyze immune and pathogen repertoires and their interplay within the host, bringing together key international players in the field and Singapore-based researchers and clinician-scientists. The focus was in particular on the application of these technologies for the improvement of patient diagnosis, prognosis and treatment, and for other broad public health outcomes. The presentations by scientists and clinicians showed the potential of deep sequencing technology to capture the coevolution of adaptive immunity and pathogens. For clinical applications, some key challenges remain, such as the long turnaround time and relatively high cost of deep sequencing for pathogen identification and characterization and the lack of international standardization in immune repertoire analysis.
RESUMO
The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach-single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.
Assuntos
Blastocisto/enzimologia , Metilação de DNA , Enzimas de Restrição do DNA/metabolismo , Análise de Célula Única/métodos , 5-Metilcitosina/metabolismo , Animais , Blastocisto/química , Blastômeros/química , Blastômeros/enzimologia , Epigênese Genética , Feminino , Impressão Genômica , CamundongosRESUMO
Circulating tumour DNA (ctDNA) has the potential to be a specific biomarker for the monitoring of tumours in patients with colorectal cancer (CRC). Here, our aim was to develop a personalised surveillance strategy to monitor the clinical course of CRC after surgery. We developed patient-specific ctDNA assays based on multiplexed detection of somatic mutations identified from patient primary tumours, and applied them to detect ctDNA in 44 CRC patients, analysing a total of 260 plasma samples. We found that ctDNA detection correlated with clinical events - it is detectable in pre-operative but not post-operative plasma, and also in patients with recurrent CRC. We also detected ctDNA in 11 out of 15 cases at or before clinical or radiological recurrence of CRC, indicating the potential of our assay for early detection of metastasis. We further present data from a patient with multiple primary cancers to demonstrate the specificity of our assays to distinguish between CRC recurrence and a second primary cancer. Our approach can complement current methods for surveillance of CRC by adding an individualised biological component, allowing us not only to point to the presence of residual or recurrent disease, but also attribute it to the original cancer.
Assuntos
Biomarcadores Tumorais , DNA Tumoral Circulante , Neoplasias Colorretais/genética , DNA de Neoplasias , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Humanos , Reação em Cadeia da Polimerase Multiplex , Mutação , Período Pós-Operatório , Recidiva , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do Tratamento , Fluxo de TrabalhoRESUMO
This protocol details a method for measuring the DNA methylation state of multiple target sites in single cells, otherwise known as single-cell restriction analysis of methylation (SCRAM). The basic steps include isolating and lysing single cells, digesting genomic DNA with a methylation-sensitive restriction endonuclease (MSRE) and amplification of multiple targets by two rounds of PCR to determine the methylation status of target sites. The method can reliably and accurately detect the methylation status of multiple target sites in each single cell, and it can be completed in a relatively short time (<2 d) at low cost. Consequently, the method may be preferable over whole-genome methods in applications requiring highly reliable and cost-effective coverage of specific target sites in all cells from a sample and in cases when the DNA methylation states of single CpG sites are representative of the methylation status of corresponding regions of interest.
Assuntos
Metilação de DNA , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Célula Única/métodos , Animais , Blastômeros/citologia , Ilhas de CpG , DNA/isolamento & purificação , Impressão Genômica , Dispositivos Lab-On-A-Chip , Camundongos , Reação em Cadeia da Polimerase Multiplex/instrumentação , Oócitos/citologia , Oócitos/fisiologiaRESUMO
We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.
Assuntos
Haplótipos/genética , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Variação Genética , Hepatite B/genética , Hepatite B/virologia , Humanos , SoftwareRESUMO
Single cell techniques permit the analysis of cellular properties that are obscured by studying the average behavior of cell populations. One way to determine how gene expression contributes to phenotypic differences among cells is to combine functional analysis with transcriptional profiling of single cells. Here we describe a microfluidic device for monitoring the responses of single cells to a ligand and then collecting cells of interest for transcriptional profiling or other assays. As a test, cells from the olfactory epithelium of zebrafish were screened by calcium imaging to identify sensory neurons that were responsive to the odorant L-lysine. Single cells were subsequently recovered for transcriptional profiling by qRT-PCR. Responsive cells all expressed TRPC2 but not OMP, consistent with known properties of amino-acid sensitive olfactory neurons. The device can be adapted for other areas in biology where there is a need to sort and analyze cells based on their signaling responses.
Assuntos
Rastreamento de Células/métodos , Técnicas Analíticas Microfluídicas/instrumentação , Neurônios Receptores Olfatórios/metabolismo , Análise de Célula Única/instrumentação , Animais , Cálcio/química , Expressão Gênica/genética , Ligantes , Lisina/administração & dosagem , Técnicas Analíticas Microfluídicas/métodos , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Análise de Célula Única/métodos , Peixe-ZebraRESUMO
Epigenetic alterations are increasingly recognized as causes of human cancers and disease. These aberrations are likely to arise during genomic reprogramming in mammalian preimplantation embryos, when their epigenomes are most vulnerable. However, this process is only partially understood because of the experimental inaccessibility of early-stage embryos. Here, we introduce a methodologic advance, probing single cells for various DNA-methylation errors at multiple loci, to reveal failed maintenance of epigenetic mark results in chimeric mice, which display unpredictable phenotypes leading to developmental arrest. Yet we show that mouse pronuclear transfer can be used to ameliorate such reprogramming defects. This study not only details the epigenetic reprogramming dynamics in early mammalian embryos but also suggests diagnostic and potential future therapeutic applications.
Assuntos
Blastocisto/metabolismo , Reprogramação Celular/genética , Quimerismo , Metilação de DNA , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Deleção de Genes , Loci Gênicos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Análise de Célula Única , Proteína 28 com Motivo TripartidoRESUMO
Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.
Assuntos
Cromatografia/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas Analíticas Microfluídicas/métodosRESUMO
The sensor histidine kinase A (KinA) from Bacillus subtilis triggers a phosphorelay that activates sporulation. The antikinase KipI prevents sporulation by binding KinA and inhibiting the autophosphorylation reaction. Using neutron contrast variation, mutagenesis, and fluorescence data, we show that two KipI monomers bind via their C-domains at a conserved proline in the KinA dimerization and histidine-phosphotransfer (DHp) domain. Our crystal structure of the KipI C-domain reveals the binding motif has a distinctive hydrophobic groove formed by a five-stranded antiparallel beta-sheet; a characteristic of the cyclophilin family of proteins that bind prolines and often act as cis-trans peptidyl-prolyl isomerases. We propose that the DHp domain of KinA transmits conformational signals to regulate kinase activity via this proline-mediated interaction. Given that both KinA and KipI homologues are widespread in the bacterial kingdom, this mechanism has broad significance in bacterial signal transduction.