RESUMEN
Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Nanoporos , ARN de Hongos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Análisis de Secuencia de ARN/métodosRESUMEN
Following cell stress such as ionising radiation (IR) exposure, multiple cellular pathways are activated. We recently demonstrated that ferredoxin reductase (FDXR) has a remarkable IR-induced transcriptional responsiveness in blood. Here, we provided a first comprehensive FDXR variant profile following DNA damage. First, specific quantitative real-time polymerase chain reaction (qPCR) primers were designed to establish dose-responses for eight curated FDXR variants, all up-regulated after IR in a dose-dependent manner. The potential role of gender on the expression of these variants was tested, and neither the variants response to IR nor the background level of expression was profoundly affected; moreover, in vitro induction of inflammation temporarily counteracted IR response early after exposure. Importantly, transcriptional up-regulation of these variants was further confirmed in vivo in blood of radiotherapy patients. Full-length nanopore sequencing was performed to identify other FDXR variants and revealed the high responsiveness of FDXR-201 and FDXR-208. Moreover, FDXR-218 and FDXR-219 showed no detectable endogenous expression, but a clear detection after IR. Overall, we characterised 14 FDXR transcript variants and identified for the first time their response to DNA damage in vivo. Future studies are required to unravel the function of these splicing variants, but they already represent a new class of radiation exposure biomarkers.
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Sangre/efectos de la radiación , Neoplasias/genética , Oxidorreductasas/genética , Regulación hacia Arriba , Adulto , Empalme Alternativo , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/radioterapia , Radiación IonizanteRESUMEN
Digital production, transmission and storage have revolutionized how we access and use information but have also made archiving an increasingly complex task that requires active, continuing maintenance of digital media. This challenge has focused some interest on DNA as an attractive target for information storage because of its capacity for high-density information encoding, longevity under easily achieved conditions and proven track record as an information bearer. Previous DNA-based information storage approaches have encoded only trivial amounts of information or were not amenable to scaling-up, and used no robust error-correction and lacked examination of their cost-efficiency for large-scale information archival. Here we describe a scalable method that can reliably store more information than has been handled before. We encoded computer files totalling 739 kilobytes of hard-disk storage and with an estimated Shannon information of 5.2 × 10(6) bits into a DNA code, synthesized this DNA, sequenced it and reconstructed the original files with 100% accuracy. Theoretical analysis indicates that our DNA-based storage scheme could be scaled far beyond current global information volumes and offers a realistic technology for large-scale, long-term and infrequently accessed digital archiving. In fact, current trends in technological advances are reducing DNA synthesis costs at a pace that should make our scheme cost-effective for sub-50-year archiving within a decade.
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Archivos , ADN/química , ADN/síntesis química , Gestión de la Información/métodos , Secuencia de Bases , Computadores , ADN/economía , Gestión de la Información/economía , Datos de Secuencia Molecular , Análisis de Secuencia de ADN/economía , Biología Sintética/economía , Biología Sintética/métodosRESUMEN
High-throughput RNA sequencing is an increasingly accessible method for studying gene structure and activity on a genome-wide scale. A critical step in RNA-seq data analysis is the alignment of partial transcript reads to a reference genome sequence. To assess the performance of current mapping software, we invited developers of RNA-seq aligners to process four large human and mouse RNA-seq data sets. In total, we compared 26 mapping protocols based on 11 programs and pipelines and found major performance differences between methods on numerous benchmarks, including alignment yield, basewise accuracy, mismatch and gap placement, exon junction discovery and suitability of alignments for transcript reconstruction. We observed concordant results on real and simulated RNA-seq data, confirming the relevance of the metrics employed. Future developments in RNA-seq alignment methods would benefit from improved placement of multimapped reads, balanced utilization of existing gene annotation and a reduced false discovery rate for splice junctions.
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Empalme del ARN , Alineación de Secuencia/métodos , Análisis de Secuencia de ARN/métodos , Animales , Mapeo Cromosómico/métodos , Biología Computacional/métodos , Exones , Reacciones Falso Positivas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Células K562 , Ratones , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Intra-tumour genetic heterogeneity is the result of ongoing evolutionary change within each cancer. The expansion of genetically distinct sub-clonal populations may explain the emergence of drug resistance, and if so, would have prognostic and predictive utility. However, methods for objectively quantifying tumour heterogeneity have been missing and are particularly difficult to establish in cancers where predominant copy number variation prevents accurate phylogenetic reconstruction owing to horizontal dependencies caused by long and cascading genomic rearrangements. To address these challenges, we present MEDICC, a method for phylogenetic reconstruction and heterogeneity quantification based on a Minimum Event Distance for Intra-tumour Copy-number Comparisons. Using a transducer-based pairwise comparison function, we determine optimal phasing of major and minor alleles, as well as evolutionary distances between samples, and are able to reconstruct ancestral genomes. Rigorous simulations and an extensive clinical study show the power of our method, which outperforms state-of-the-art competitors in reconstruction accuracy, and additionally allows unbiased numerical quantification of tumour heterogeneity. Accurate quantification and evolutionary inference are essential to understand the functional consequences of tumour heterogeneity. The MEDICC algorithms are independent of the experimental techniques used and are applicable to both next-generation sequencing and array CGH data.
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Neoplasias/clasificación , Filogenia , Alelos , Evolución Biológica , Humanos , Neoplasias/patologíaRESUMEN
The 1000 Genomes Project data provides a natural background dataset for amino acid germline mutations in humans. Since the direction of mutation is known, the amino acid exchange matrix generated from the observed nucleotide variants is asymmetric and the mutabilities of the different amino acids are very different. These differences predominantly reflect preferences for nucleotide mutations in the DNA (especially the high mutation rate of the CpG dinucleotide, which makes arginine mutability very much higher than other amino acids) rather than selection imposed by protein structure constraints, although there is evidence for the latter as well. The variants occur predominantly on the surface of proteins (82%), with a slight preference for sites which are more exposed and less well conserved than random. Mutations to functional residues occur about half as often as expected by chance. The disease-associated amino acid variant distributions in OMIM are radically different from those expected on the basis of the 1000 Genomes dataset. The disease-associated variants preferentially occur in more conserved sites, compared to 1000 Genomes mutations. Many of the amino acid exchange profiles appear to exhibit an anti-correlation, with common exchanges in one dataset being rare in the other. Disease-associated variants exhibit more extreme differences in amino acid size and hydrophobicity. More modelling of the mutational processes at the nucleotide level is needed, but these observations should contribute to an improved prediction of the effects of specific variants in humans.
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Aminoácidos/genética , Bases de Datos Genéticas , Genoma Humano , Humanos , Mutación , Proteínas/química , Proteínas/genéticaRESUMEN
In the era of ubiquitous high-throughput sequencing studies, there is a growing need for analysis tools that are not just performant but also comprehensive and user-friendly enough to cater to both novice and advanced users. This article introduces SeqKit2, the next iteration of the widely used sequence analysis tool SeqKit, featuring expanded functionality, performance optimizations, and support for additional compression methods. Retaining a pragmatic subcommand architecture, SeqKit2 represents substantial enhancement through the inclusion of 19 additional subcommands, expanding its overall repertoire to a total of 38 in eight categories. The new subcommands add functionality such as amplicon processing and robust, error-tolerant parsing of sequence records. In addition, three subcommands designed for real-time analysis are added for periodic monitoring of properties of FASTQ and Binary Alignment/Map alignment records and real-time streaming from multiple sequence files. The performance of SeqKit2 is benchmarked against the old version of SeqKit, Bioawk, Seqtk, and SeqFu tools. SeqKit2 consistently outperforms its predecessor, albeit with marginally higher memory usage, while maintaining competitive runtimes against other tools. With its broad functionality, proven usability, and ongoing development driven by user feedback, we hope that bioinformaticians will find SeqKit2 useful as a "Swiss army knife" of sequence and alignment processing-equally adept at facilitating ad hoc analyses and seamlessly integrating into larger pipelines.
RESUMEN
Biological age is typically estimated using biomarkers whose states have been observed to correlate with chronological age. A persistent limitation of such aging clocks is that it is difficult to establish how the biomarker states are related to the mechanisms of aging. Somatic mutations could potentially form the basis for a more fundamental aging clock since the mutations are both markers and drivers of aging and have a natural timescale. Cell lineage trees inferred from these mutations reflect the somatic evolutionary process, and thus, it has been conjectured, the aging status of the body. Such a timer has been impractical thus far, however, because detection of somatic variants in single cells presents a significant technological challenge. Here, we show that somatic mutations detected using single-cell RNA sequencing (scRNA-seq) from thousands of cells can be used to construct a cell lineage tree whose structure correlates with chronological age. De novo single-nucleotide variants (SNVs) are detected in human peripheral blood mononuclear cells using a modified protocol. A default model based on penalized multiple regression of chronological age on 31 metrics characterizing the phylogenetic tree gives a Pearson correlation of 0.81 and a median absolute error of ~4 years between predicted and chronological ages. Testing of the model on a public scRNA-seq dataset yields a Pearson correlation of 0.85. In addition, cell tree age predictions are found to be better predictors of certain clinical biomarkers than chronological age alone, for instance glucose, albumin levels, and leukocyte count. The geometry of the cell lineage tree records the structure of somatic evolution in the individual and represents a new modality of aging timer. In addition to providing a numerical estimate of "cell tree age," it unveils a temporal history of the aging process, revealing how clonal structure evolves over life span. Cell Tree Rings complements existing aging clocks and may help reduce the current uncertainty in the assessment of geroprotective trials.
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Envejecimiento , Leucocitos Mononucleares , Humanos , Filogenia , Envejecimiento/genética , Longevidad , BiomarcadoresRESUMEN
The blood cells, or hemocytes, in Drosophila participate in the immune response through the production of antimicrobial peptides, the phagocytosis of bacteria, and the encapsulation of larger foreign particles such as parasitic eggs; these immune reactions are mediated by phylogenetically conserved mechanisms. The encapsulation reaction is analogous to the formation of granuloma in vertebrates, and is mediated by large specialized cells, the lamellocytes. The origin of the lamellocytes has not been formally established, although it has been suggested that they are derived from the lymph gland, which is generally considered to be the main hematopoietic organ in the Drosophila larva. However, it was recently observed that a subepidermal population of sessile blood cells is released into the circulation in response to a parasitoid wasp infection. We set out to analyze this phenomenon systematically. As a result, we define the sessile hemocytes as a novel hematopoietic compartment, and the main source of lamellocytes.
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Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/inmunología , Hematopoyesis , Hemocitos/citología , Animales , Recuento de Células , Diferenciación Celular , Separación Celular , Drosophila melanogaster/citología , Proteínas Fluorescentes Verdes/metabolismo , Hemocitos/trasplante , Inmunidad , Larva/citología , Larva/inmunología , Larva/parasitología , Fenotipo , Factores de TiempoRESUMEN
BACKGROUND: The Monte Carlo simulation of sequence evolution is routinely used to assess the performance of phylogenetic inference methods and sequence alignment algorithms. Progress in the field of molecular evolution fuels the need for more realistic and hence more complex simulations, adapted to particular situations, yet current software makes unreasonable assumptions such as homogeneous substitution dynamics or a uniform distribution of indels across the simulated sequences. This calls for an extensible simulation framework written in a high-level functional language, offering new functionality and making it easy to incorporate further complexity. RESULTS: PhyloSim is an extensible framework for the Monte Carlo simulation of sequence evolution, written in R, using the Gillespie algorithm to integrate the actions of many concurrent processes such as substitutions, insertions and deletions. Uniquely among sequence simulation tools, PhyloSim can simulate arbitrarily complex patterns of rate variation and multiple indel processes, and allows for the incorporation of selective constraints on indel events. User-defined complex patterns of mutation and selection can be easily integrated into simulations, allowing PhyloSim to be adapted to specific needs. CONCLUSIONS: Close integration with R and the wide range of features implemented offer unmatched flexibility, making it possible to simulate sequence evolution under a wide range of realistic settings. We believe that PhyloSim will be useful to future studies involving simulated alignments.
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Modelos Genéticos , Método de Montecarlo , Algoritmos , Animales , Secuencia de Bases , Simulación por Computador , Evolución Molecular , Humanos , Cómputos Matemáticos , Filogenia , Programas InformáticosRESUMEN
In the event of a large-scale event leading to acute ionizing radiation exposure, high-throughput methods would be required to assess individual dose estimates for triage purposes. Blood-based gene expression is a broad source of biomarkers of radiation exposure which have great potential for providing rapid dose estimates for a large population. Time is a crucial component in radiological emergencies and the shipment of blood samples to relevant laboratories presents a concern. In this study, we performed nanopore sequencing analysis to determine if the technology can be used to detect radiation-inducible genes in human peripheral blood mononuclear cells (PBMCs). The technology offers not only long-read sequencing but also a portable device which can overcome issues involving sample shipment, and provide faster results. For this goal, blood from nine healthy volunteers was 2 Gy ex vivo X irradiated. After PBMC isolation, irradiated samples were incubated along with the controls for 24 h at 37°C. RNA was extracted, poly(A)+ enriched and reverse-transcribed before sequencing. The data generated was analyzed using a Snakemake pipeline modified to handle paired samples. The sequencing analysis identified a radiation signature consisting of 46 differentially expressed genes (DEGs) which included 41 protein-coding genes, a long non-coding RNA and four pseudogenes, five of which have been identified as radiation-responsive transcripts for the first time. The genes in which transcriptional expression is most significantly modified after radiation exposure were APOBEC3H and FDXR, presenting a 25- and 28-fold change on average, respectively. These levels of transcriptional response were comparable to results we obtained by quantitative polymerase chain reaction (qPCR) analysis. In vivo exposure analyses showed a transcriptional radioresponse at 24 h postirradiation for both genes together with a strong dose-dependent response in blood irradiated ex vivo. Finally, extrapolating from the data we obtained, the minimum sequencing time required to detect an irradiated sample using APOBEC3H transcripts would be less than 3 min for a total of 50,000 reads. Future improvements, in sample processing and bioinformatic pipeline for specific radiation-responsive transcript identification, will allow the provision of a portable, rapid, real-time biodosimetry platform based on this new sequencing technology. In summary, our data show that nanopore sequencing can identify radiation-responsive genes and can also be used for identification of new transcripts.
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Sangre/metabolismo , Sangre/efectos de la radiación , Secuenciación de Nanoporos , Exposición a la Radiación/efectos adversos , Transcripción Genética/efectos de la radiación , Transcriptoma/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Genómica , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/efectos de la radiaciónRESUMEN
The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response.
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Evolución Molecular , Genes de Insecto , Familia de Multigenes , Secuencias Repetitivas de Aminoácido , Secuencias de Aminoácidos , Animales , Anopheles/genética , Abejas/genética , Drosophila/genética , Filogenia , Alineación de Secuencia , Tribolium/genéticaRESUMEN
BACKGROUND: The models developed to characterize the evolution of multigene families (such as the birth-and-death and the concerted models) have also been applied on the level of sequence repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the evolution of gene families and also sequence repeats in the light of these models. The characterization of the gene family evolution in view of the evolutionary models is done by the evaluation of the clustering of the sequences with the originating loci in mind. As the locus represents positional information, it is straightforward that in the case of the repeats the exact position in the sequence should be used, as the simple numbering according to repeat order can be misleading. RESULTS: We have developed a novel rapid visual approach to study repeat evolution, that takes into account the exact repeat position in a sequence. The "pairwise repeat homology diagram" visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script (t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences. CONCLUSION: Although pairwise repeat homology diagrams do not carry all the information provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution. We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This method can be particularly useful in cases of large datasets (such as large gene families), as the command line interface makes it possible to automate the generation of pairwise repeat homology diagrams with the aid of scripts.
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Evolución Biológica , Mapeo Cromosómico/métodos , Evolución Molecular , Familia de Multigenes/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Algoritmos , Secuencia de Bases , Análisis Mutacional de ADN , Datos de Secuencia Molecular , Reconocimiento de Normas Patrones Automatizadas , FilogeniaRESUMEN
The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.
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Antígenos de Grupos Sanguíneos/genética , Membrana Eritrocítica/metabolismo , Eritrocitos/inmunología , Eliminación de Gen , Homocigoto , Proteínas de la Membrana/genética , Sitios de Carácter Cuantitativo , Alelos , Animales , Biomarcadores/metabolismo , Antígenos de Grupos Sanguíneos/inmunología , Antígenos de Grupos Sanguíneos/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Eritrocitos/metabolismo , Eritrocitos/patología , Exoma/genética , Femenino , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Isoanticuerpos/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Embarazo , Pez Cebra/genéticaRESUMEN
The rise of Next Generation Sequencing (NGS) technologies has transformed de novo genome sequencing into an accessible research tool, but obtaining high quality eukaryotic genome assemblies remains a challenge, mostly due to the abundance of repetitive elements. These also make it difficult to study nucleotide polymorphism in repetitive regions, including certain types of structural variations. One solution proposed for resolving such regions is Sequence Assembly aided by Mutagenesis (SAM), which relies on the fact that introducing enough random mutations breaks the repetitive structure, making assembly possible. Sequencing many different mutated copies permits the sequence of the repetitive region to be inferred by consensus methods. However, this approach relies on molecular cloning in order to isolate and amplify individual mutant copies, making it hard to scale-up the approach for use in conjunction with high-throughput sequencing technologies. To address this problem, we propose NG-SAM, a modified version of the SAM protocol that relies on PCR and dilution steps only, coupled to a NGS workflow. NG-SAM therefore has the potential to be scaled-up, e.g. using emerging microfluidics technologies. We built a realistic simulation pipeline to study the feasibility of NG-SAM, and our results suggest that under appropriate experimental conditions the approach might be successfully put into practice. Moreover, our simulations suggest that NG-SAM is capable of reconstructing robustly a wide range of potential target sequences of varying lengths and repetitive structures.
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Genoma/genética , Mutagénesis , Secuencias Repetitivas de Ácidos Nucleicos/genética , Análisis de Secuencia de ADN/métodos , Animales , Secuencia de Bases , Simulación por Computador , Estudios de Factibilidad , Genómica/métodos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Reproducibilidad de los ResultadosRESUMEN
Gray platelet syndrome (GPS) is a predominantly recessive platelet disorder that is characterized by mild thrombocytopenia with large platelets and a paucity of α-granules; these abnormalities cause mostly moderate but in rare cases severe bleeding. We sequenced the exomes of four unrelated individuals and identified NBEAL2 as the causative gene; it has no previously known function but is a member of a gene family that is involved in granule development. Silencing of nbeal2 in zebrafish abrogated thrombocyte formation.
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Plaquetas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Síndrome de Plaquetas Grises/genética , Proteínas del Tejido Nervioso/genética , Vesículas Secretoras/metabolismo , Adulto , Anciano , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Plaquetas/patología , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Linaje , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Adulto Joven , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
The Nimrod gene superfamily is an important component of the innate immune response. The majority of its member genes are located in close proximity within the Drosophila melanogaster genome and they lie in a larger conserved cluster ("Nimrod cluster"), made up of non-related groups (families, superfamilies) of genes. This cluster has been a part of the Arthropod genomes for about 300-350 million years. The available data suggest that the Nimrod cluster is a functional module of the insect innate immune response.