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BACKGROUND: Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50 bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. RESULTS: In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools-FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus-ranged from 5.8-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. CONCLUSIONS: We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently existing software. However, methods for identification of genomic variants using scRNA-seq data can be still improved.
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Fibroblastos , Polimorfismo de Nucleótido Simple , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Programas Informáticos , Secuenciación del ExomaRESUMEN
OBJECTIVE: Patients with rheumatic diseases such as rheumatoid arthritis (RA) and lupus have increased risk of infection and are treated with medications that may increase this risk yet are also hypothesized to help treat COVID-19. We set out to understand how the COVID-19 pandemic has impacted the lives of these patients in the United States. METHODS: Participants in a US-wide longitudinal observational registry responded to a supplemental COVID-19 questionnaire by e-mail on March 25, 2020, about their symptoms, COVID-19 testing, health care changes, and related experiences during the prior 2 weeks. Analysis compared responses by diagnosis, disease activity, and new onset of symptoms. Qualitative analysis was conducted on optional free-text comment fields. RESULTS: Of the 7061 participants invited to participate, 530 responded, with RA as the most frequent diagnosis (61%). Eleven participants met COVID-19 screening criteria, of whom two sought testing unsuccessfully. Six others sought testing, three of whom were successful, and all test results were negative. Not quite half of participants (42%) reported a change to their care in the prior 2 weeks. Qualitative analysis revealed four key themes: emotions in response to the pandemic, perceptions of risks from immunosuppressive medications, protective measures to reduce risk of COVID-19 infection, and disruptions in accessing rheumatic disease medications, including hydroxychloroquine. CONCLUSION: After 2 weeks, many participants with rheumatic diseases already had important changes to their health care, with many altering medications without professional consultation or because of hydroxychloroquine shortage. As evidence accumulates on the effectiveness of potential COVID-19 treatments, effort is needed to safeguard access to established treatments for rheumatic diseases.
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Exome sequencing of two sisters with congenital cataracts, short stature, and white matter changes identified compound heterozygous variants in the PISD gene, encoding the phosphatidylserine decarboxylase enzyme that converts phosphatidylserine to phosphatidylethanolamine (PE) in the inner mitochondrial membrane (IMM). Decreased conversion of phosphatidylserine to PE in patient fibroblasts is consistent with impaired phosphatidylserine decarboxylase (PISD) enzyme activity. Meanwhile, as evidence for mitochondrial dysfunction, patient fibroblasts exhibited more fragmented mitochondrial networks, enlarged lysosomes, decreased maximal oxygen consumption rates, and increased sensitivity to 2-deoxyglucose. Moreover, treatment with lyso-PE, which can replenish the mitochondrial pool of PE, and genetic complementation restored mitochondrial and lysosome morphology in patient fibroblasts. Functional characterization of the PISD variants demonstrates that the maternal variant causes an alternative splice product. Meanwhile, the paternal variant impairs autocatalytic self-processing of the PISD protein required for its activity. Finally, evidence for impaired activity of mitochondrial IMM proteases suggests an explanation as to why the phenotypes of these PISD patients resemble recently described "mitochondrial chaperonopathies." Collectively, these findings demonstrate that PISD is a novel mitochondrial disease gene.
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Carboxiliasas/genética , Catarata/genética , Enfermedades Mitocondriales/enzimología , Anomalías Musculoesqueléticas/genética , Sustancia Blanca/patología , Adulto , Carboxiliasas/metabolismo , Femenino , Fibroblastos/metabolismo , Genes Mitocondriales/genética , Células HEK293 , Homeostasis/genética , Humanos , Mitocondrias/enzimología , Enfermedades Mitocondriales/sangre , Enfermedades Mitocondriales/patología , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Fenotipo , Sitios de Empalme de ARN/genética , Saccharomyces cerevisiae/enzimología , Transfección , Secuenciación del ExomaRESUMEN
Glioblastoma (GBM) is the most common and the deadliest type of primary brain tumor, with a median survival time of only 15 months despite aggressive treatment. Although most patients have an extremely poor prognosis, a relatively small number of patients survive far beyond the median survival time. Investigation of these exceptional responders has sparked a great deal of interest and is becoming an important focus in the field of cancer research. To investigate the molecular differences between typical and exceptional responders in GBM, comparative analyses of somatic mutations, copy number, methylation, and gene expression datasets from The Cancer Genome Atlas were performed, and the results of these analyses were integrated via gene ontology and pathway analyses to assess the functional significance of the differential aberrations. Less severe copy number loss of CDKN2A, lower expression of CXCL8, and FLG mutations are all associated with an exceptional response. Typical responders are characterized by upregulation of NF-κB signaling and of pro-inflammatory cytokines, while exceptional responders are characterized by upregulation of Alzheimer's and Parkinson's disease pathways as well as of genes involved in synaptic transmission. The upregulated pathways and processes in typical responders are consistently associated with more aggressive tumor phenotypes, while those in the exceptional responders suggest a retained ability in tumor cells to undergo cell death in response to treatment. With the upcoming launch of the National Cancer Institute's Exceptional Responders Initiative, similar studies with much larger sample sizes will likely become possible, hopefully providing even more insight into the molecular differences between typical and exceptional responders.
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T-cell clonality of peripheral T-cell lymphoma (PTCL) is routinely evaluated with a PCR-based method using genomic DNA. However, there are limitations with this approach. The purpose of this study was to determine the utility of RNA-seq for assessing T-cell clonality and T-cell antigen receptor (TCR) repertoire of the neoplastic T-cells in 108 PTCL samples. TCR transcripts, including complementarity-determining region 3 (CDR3) sequences, were assessed. In normal T cells, the CDR3 sequences were extremely diverse, without any clonotype representing more than 2% of the overall TCR population. Dominant clones could be identified in 65 out of 76 PTCL cases (86%) with adequate TCR transcript expression. In monoclonal cases, the dominant clone varied between 11% and 99% of TCRß transcripts. No unique Vα or Vß usage was observed. Small T-cell clones were often observed in T- and NK-cell tumors in a percentage higher than observed in reactive conditions. γ chain expression was very low in tumors expressing TCRαß, but its expression level was high and clonality was detected in a TCRγδ expressing tumor. NK cell lymphoma (NKCL) did not express significant levels of TCR Vß or Vγ genes. RNA-seq is a useful tool for detecting and characterizing clonal TCR rearrangements in PTCL.
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Evolución Clonal/genética , Linfoma de Células T Periférico/genética , Receptores de Antígenos de Linfocitos T/genética , Biología Computacional/métodos , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Linfoma de Células T Periférico/patología , Mutación , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1003064.].
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The proton pathway of [FeFe]-hydrogenase is essential for enzymatic H2 production and oxidation and is composed of four residues and a water molecule. A computational analysis of this pathway in the [FeFe]-hydrogenase from Clostridium pasteurianum revealed that the solvent-exposed residue of the pathway (Glu282) forms hydrogen bonds to two residues outside of the pathway (Arg286 and Ser320), implying that these residues could function in regulating proton transfer. In this study, we show that substituting Arg286 with leucine eliminates hydrogen bonding with Glu282 and results in an â¼3-fold enhancement of H2 production activity when methyl viologen is used as an electron donor, suggesting that Arg286 may help control the rate of proton delivery. In contrast, substitution of Ser320 with alanine reduces the rate â¼5-fold, implying that it either acts as a member of the pathway or influences Glu282 to permit proton transfer. Interestingly, quantum mechanics/molecular mechanics and molecular dynamics calculations indicate that Ser320 does not play a structural role or indirectly influence the barrier for proton movement at the entrance of the channel. Rather, it may act as an additional proton acceptor for the pathway or serve in a regulatory role. While further studies are needed to elucidate the role of Ser320, collectively these data provide insights into the complex proton transport process.
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Aminoácidos/química , Proteínas Bacterianas/metabolismo , Clostridium/enzimología , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Proteínas Mutantes/metabolismo , Mutación/genética , Protones , Aminoácidos/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Hidrogenasas/química , Hidrogenasas/genética , Transporte Iónico , Hierro/metabolismo , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/genética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genéticaRESUMEN
Natural killer/T-cell lymphoma (NKTCL) is a rare, aggressive form of non-Hodgkin lymphoma that is generally incurable at more advanced stages with systemic involvement. Clonal diagnostic markers (eg, unique T- or B-cell receptor rearrangements) are not available for NKTCLs. Killer cell immunoglobulin like receptors (KIRs) are a family of type I transmembrane glycoproteins involved in the inhibition or activation of NK cells. A restricted expression profile of KIRs has been proposed as clonal markers of NK-cell proliferations. Here we evaluated the transcription profile of all KIR family genes and C-type lectin receptor genes using RNA sequencing on NKTCL cases (n = 17) and NK-cell lines (n = 3). The expression of all KIRs tended to be markedly reduced or absent in NKTCL, except for the KIR family member killer Ig-like receptor 2DL4 (KIR2DL4; alias CD158D), which was selectively overexpressed in the majority (59%) of cases. No specific expression pattern was observed for C-type lectin receptors. KIR2DL4 is an unusual member of the KIR family that recognizes human leukocyte antigen G and mediates NK-cell activation through inducing proliferation and survival pathways such as AKT and NF-κB. Stable knockdown of KIR2DL4 in two malignant NK-cell lines with high KIR2DL4 expression significantly reduced cell growth. Selective overexpression of KIR2DL4 and down-regulation of inhibitory KIRs may contribute to NKTCL pathogenesis.
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Biomarcadores de Tumor/análisis , Linfoma Extranodal de Células NK-T/metabolismo , Receptores KIR/biosíntesis , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores KIR/análisis , Transcriptoma , Células Tumorales CultivadasRESUMEN
RATIONALE: Hydrogenases catalyze the reversible formation of H2 from electrons and protons with high efficiency. Understanding the relationships between H2 production, H2 uptake, and H2-H2O exchange can provide insight into the metabolism of microbial communities in which H2 is an essential component in energy cycling. METHODS: We used stable H isotopes (1H and 2H) to probe the isotope effects associated with three [FeFe]-hydrogenases and three [NiFe]-hydrogenases. RESULTS: All six hydrogenases displayed fractionation factors for H2 formation that were significantly less than 1, producing H2 that was severely depleted in 2H relative to the substrate, water. Consistent with differences in their active site structure, the fractionation factors for each class appear to cluster, with the three [NiFe]-hydrogenases (α = 0.270.40) generally having smaller values than the three [FeFe]-hydrogenases (α = 0.410.55). We also obtained isotopic fractionation factors associated with H2 uptake and H2-H2O exchange under conditions similar to those utilized for H2 production, providing a more complete picture of the reactions catalyzed by hydrogenases. CONCLUSIONS: The fractionation factors determined in our studies can be used as signatures for different hydrogenases to probe their activity under different growth conditions and to ascertain which hydrogenases are most responsible for H2 production and/or uptake in complex microbial communities.
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Hidrógeno/química , Hidrogenasas/química , Proteínas Hierro-Azufre/química , Fraccionamiento Químico , Chlamydomonas reinhardtii/enzimología , Clostridium/enzimología , Deuterio/química , Shewanella/enzimologíaRESUMEN
BACKGROUND: Exome sequencing has been utilized to identify genetic variants associated with disease in humans. Identification of loss-of-function mutations with exome sequencing in rhesus macaques (Macaca mulatta) could lead to valuable animal models of genetic disease. Attempts have been made to identify variants in rhesus macaques by aligning exome data against the rheMac2 draft genome. However, such efforts have been impaired due to the incompleteness and annotation errors associated with rheMac2. We wished to determine whether aligning exome reads against our new, improved rhesus genome, MacaM, could be used to identify high impact, loss-of-function mutations in rhesus macaques that would be relevant to human disease. RESULTS: We compared alignments of exome reads from four rhesus macaques, the reference animal and three unrelated animals, against rheMac2 and MacaM. Substantially more reads aligned against MacaM than rheMac2. We followed the Broad Institute's Best Practice guidelines for variant discovery which utilizes the Genome Analysis Toolkit to identify high impact mutations. When rheMac2 was used as the reference genome, a large number of apparent false positives were identified. When MacaM was used as the reference genome, the number of false positives was greatly reduced. After examining the variant analyses conducted with MacaM as reference genome, we identified two putative loss-of-function mutations, in the heterozygous state, in genes related to human health. Sanger sequencing confirmed the presence of these mutations. We followed the transmission of one of these mutations (in the butyrylthiocholine gene) through three generations of rhesus macaques. Further, we demonstrated a functional decrease in butyrylthiocholinesterase activity similar to that observed in human heterozygotes with loss-of-function mutations in the same gene. CONCLUSIONS: The new MacaM genome can be effectively utilized to identify loss-of-function mutations in rhesus macaques without generating a high level of false positives. In some cases, heterozygotes may be immediately useful as models of human disease. For diseases where homozygous mutants are needed, directed breeding of loss-of-function heterozygous animals could be used to create rhesus macaque models of human genetic disease. The approach we describe here could be applied to other mammals, but only if their genomes have been improved beyond draft status.
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Análisis Mutacional de ADN , Exoma , Macaca mulatta/genética , Animales , Modelos Animales de Enfermedad , Heterocigoto , Humanos , Mutación , Alineación de SecuenciaRESUMEN
High-throughput sequencing, especially of exomes, is a popular diagnostic tool, but it is difficult to determine which tools are the best at analyzing this data. In this study, we use the NIST Genome in a Bottle results as a novel resource for validation of our exome analysis pipeline. We use six different aligners and five different variant callers to determine which pipeline, of the 30 total, performs the best on a human exome that was used to help generate the list of variants detected by the Genome in a Bottle Consortium. Of these 30 pipelines, we found that Novoalign in conjunction with GATK UnifiedGenotyper exhibited the highest sensitivity while maintaining a low number of false positives for SNVs. However, it is apparent that indels are still difficult for any pipeline to handle with none of the tools achieving an average sensitivity higher than 33% or a Positive Predictive Value (PPV) higher than 53%. Lastly, as expected, it was found that aligners can play as vital a role in variant detection as variant callers themselves.
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Exoma/genética , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Algoritmos , Humanos , Mutación INDEL , Polimorfismo de Nucleótido SimpleRESUMEN
Hydrogen gas functions as a key component in the metabolism of a wide variety of microorganisms, often acting as either a fermentative end-product or an energy source. The number of organisms reported to utilize hydrogen continues to grow, contributing to and expanding our knowledge of biological hydrogen processes. Here we demonstrate that Volvox carteri f. nagariensis, a multicellular green alga with differentiated cells, evolves H2 both when supplied with an abiotic electron donor and under physiological conditions. The genome of Volvox carteri contains two genes encoding putative [FeFe]-hydrogenases (HYDA1 and HYDA2), and the transcripts for these genes accumulate under anaerobic conditions. The HYDA1 and HYDA2 gene products were cloned, expressed, and purified, and both are functional [FeFe]-hydrogenases. Additionally, within the genome the HYDA1 and HYDA2 genes cluster with two putative genes which encode hydrogenase maturation proteins. This gene cluster resembles operon-like structures found within bacterial genomes and may provide further insight into evolutionary relationships between bacterial and algal [FeFe]-hydrogenase genes.
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Hidrógeno/metabolismo , Volvox/metabolismo , Proteínas Algáceas/química , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Perfilación de la Expresión Génica , Orden Génico , Hidrogenasas/química , Hidrogenasas/genética , Hidrogenasas/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Alineación de Secuencia , Volvox/genéticaRESUMEN
Lymphomas arising from NK or γδ-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n=51), γδ-T-cell lymphomas (n=43) and their cell lines (n=9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of γδ-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylated protein and a growth advantage to transduced cell lines or normal NK cells. Growth-promoting activity of the mutants can be partially inhibited by a JAK1/2 inhibitor. Molecular modelling and surface plasmon resonance measurements of the N642H mutant indicate a marked increase in binding affinity of the phosphotyrosine-Y699 with the mutant histidine. This is associated with the prolonged persistence of the mutant phosphoSTAT5B and marked increase of binding to target sites. Our findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy.
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Regulación Neoplásica de la Expresión Génica , Células Asesinas Naturales/citología , Linfoma de Células T/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT5/genética , Subgrupos de Linfocitos T/citología , Sitios de Unión , Células HEK293 , Histidina/química , Humanos , Interleucina-2/metabolismo , Janus Quinasa 1/metabolismo , Mutación , Fosfotirosina/química , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismoRESUMEN
BACKGROUND: The rhesus macaque (Macaca mulatta) is a key species for advancing biomedical research. Like all draft mammalian genomes, the draft rhesus assembly (rheMac2) has gaps, sequencing errors and misassemblies that have prevented automated annotation pipelines from functioning correctly. Another rhesus macaque assembly, CR_1.0, is also available but is substantially more fragmented than rheMac2 with smaller contigs and scaffolds. Annotations for these two assemblies are limited in completeness and accuracy. High quality assembly and annotation files are required for a wide range of studies including expression, genetic and evolutionary analyses. RESULTS: We report a new de novo assembly of the rhesus macaque genome (MacaM) that incorporates both the original Sanger sequences used to assemble rheMac2 and new Illumina sequences from the same animal. MacaM has a weighted average (N50) contig size of 64 kilobases, more than twice the size of the rheMac2 assembly and almost five times the size of the CR_1.0 assembly. The MacaM chromosome assembly incorporates information from previously unutilized mapping data and preliminary annotation of scaffolds. Independent assessment of the assemblies using Ion Torrent read alignments indicates that MacaM is more complete and accurate than rheMac2 and CR_1.0. We assembled messenger RNA sequences from several rhesus tissues into transcripts which allowed us to identify a total of 11,712 complete proteins representing 9,524 distinct genes. Using a combination of our assembled rhesus macaque transcripts and human transcripts, we annotated 18,757 transcripts and 16,050 genes with complete coding sequences in the MacaM assembly. Further, we demonstrate that the new annotations provide greatly improved accuracy as compared to the current annotations of rheMac2. Finally, we show that the MacaM genome provides an accurate resource for alignment of reads produced by RNA sequence expression studies. CONCLUSIONS: The MacaM assembly and annotation files provide a substantially more complete and accurate representation of the rhesus macaque genome than rheMac2 or CR_1.0 and will serve as an important resource for investigators conducting next-generation sequencing studies with nonhuman primates. REVIEWERS: This article was reviewed by Dr. Lutz Walter, Dr. Soojin Yi and Dr. Kateryna Makova.
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Genoma , Macaca mulatta/genética , Secuencia de Aminoácidos , Animales , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Alineación de SecuenciaRESUMEN
HACE1 is an E3 ubiquitin ligase located in 6q21, the genomic region frequently deleted in natural killer (NK) cell malignancies. Here, we report HACE1 as a candidate tumor suppressor gene silenced through a combination of deletion and cytosine phosphate guanine island hypermethylation. We detected deletion of HACE1 in malignant NK cell lines (6 of 9, 67%) and primary biopsies (5 of 15, 33%) by quantitative PCR, with most of the specimen showing cytosine phosphate guanine island hypermethylation in the remaining allele, leading to low mRNA transcription. The ectopic expression of HACE1 in an HACE1-null NK cell line led to apoptosis and G2/M cell cycle arrest. Moreover, HACE1 expression was up-regulated in IL-2-activated normal NK cells and NK cells cocultured with an engineered NK cell target, K562 Clone 9.mbIL21, suggesting its role in the regulation of NK cell homeostasis. In conclusion, HACE1 is another potent tumor suppressor gene located within the 6q21 region, and loss of function of multiple tumor suppressor genes within 6q21 may be a critical determinant of NK cell lymphomagenesis.
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Células Asesinas Naturales/metabolismo , Linfoma no Hodgkin/genética , Ubiquitina-Proteína Ligasas/genética , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Técnicas de Cocultivo , Islas de CpG/genética , Metilación de ADN , Análisis Mutacional de ADN/métodos , ADN de Neoplasias/genética , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genes Supresores de Tumor , Humanos , Interleucina-2/inmunología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Activación de Linfocitos/genética , Linfoma no Hodgkin/metabolismo , Linfoma no Hodgkin/patología , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas/biosíntesis , Regulación hacia Arriba/genéticaRESUMEN
Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.
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Genoma , Anotación de Secuencia Molecular , Estramenopilos/genética , Secuencia de Bases , Genómica , Nitrógeno/administración & dosificación , Nitrógeno/metabolismo , Análisis de Secuencia de ADN , Análisis de Secuencia de ARN/métodos , Especificidad de la Especie , Estramenopilos/crecimiento & desarrollo , Transformación GenéticaRESUMEN
H(2) generated from renewable resources holds promise as an environmentally innocuous fuel that releases only energy and water when consumed. In biotechnology, photoautotrophic oxygenic diazotrophs could produce H(2) from water and sunlight using the cells' endogenous nitrogenases. However, nitrogenases have low turnover numbers and require large amounts of ATP. [FeFe]-hydrogenases found in other organisms can have 1,000-fold higher turnover numbers and no specific requirement for ATP but are very O(2) sensitive. Certain filamentous cyanobacteria protect nitrogenase from O(2) by sequestering the enzyme within internally micro-oxic, differentiated cells called heterocysts. We heterologously expressed the [FeFe]-hydrogenase operon from Shewanella oneidensis MR-1 in Anabaena sp. strain PCC 7120 using the heterocyst-specific promoter P(hetN). Active [FeFe]-hydrogenase was detected in and could be purified from aerobically grown Anabaena sp. strain PCC 7120, but only when the organism was grown under nitrate-depleted conditions that elicited heterocyst formation. These results suggest that the heterocysts protected the [FeFe]-hydrogenase against inactivation by O(2).
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Anabaena/genética , Hidrogenasas/genética , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Shewanella/enzimología , Aerobiosis , Expresión Génica , Hidrogenasas/aislamiento & purificación , Proteínas Hierro-Azufre/aislamiento & purificación , Operón , Regiones Promotoras Genéticas , Proteínas Recombinantes/aislamiento & purificación , Shewanella/genéticaRESUMEN
[FeFe]-Hydrogenases are complex metalloproteins that catalyze the reversible reduction of protons to molecular hydrogen utilizing a unique diiron subcluster bridged to a [4Fe4S] subcluster. Extensive studies have concentrated on the nature and catalytic activity of the active site, yet relatively little information is available concerning the mechanism of proton transport that is required for this activity. Previously, structural characterization of [FeFe]-hydrogenase from Clostridium pasteurianum indicated a potential proton transport pathway involving four residues (Cys-299, Glu-279, Ser-319, and Glu-282) that connect the active site to the enzyme surface. Here, we demonstrate that substitution of any of these residues resulted in a drastic reduction in hydrogenase activity relative to the native enzyme, supporting the importance of these residues in catalysis. Inhibition studies of native and amino acid-substituted enzymes revealed that Zn(2+) specifically blocked proton transfer by binding to Glu-282, confirming the role of this residue in the identified pathway. In addition, all four of these residues are strictly conserved, suggesting that they may form a proton transport pathway that is common to all [FeFe]-hydrogenases.
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Clostridium/enzimología , Hidrogenasas/química , Hierro/química , Secuencia de Aminoácidos , Transporte Biológico , Catálisis , Ácido Glutámico/química , Proteínas Hierro-Azufre/química , Cinética , Conformación Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Protones , Homología de Secuencia de Aminoácido , Zinc/químicaRESUMEN
Like many microalgae, Chlamydomonas reinhardtii forms lipid droplets rich in triacylglycerols when nutrient deprived. To begin studying the mechanisms underlying this process, nitrogen (N) deprivation was used to induce triacylglycerol accumulation and changes in developmental programs such as gametogenesis. Comparative global analysis of transcripts under induced and noninduced conditions was applied as a first approach to studying molecular changes that promote or accompany triacylglycerol accumulation in cells encountering a new nutrient environment. Towards this goal, high-throughput sequencing technology was employed to generate large numbers of expressed sequence tags of eight biologically independent libraries, four for each condition, N replete and N deprived, allowing a statistically sound comparison of expression levels under the two tested conditions. As expected, N deprivation activated a subset of control genes involved in gametogenesis while down-regulating protein biosynthesis. Genes for components of photosynthesis were also down-regulated, with the exception of the PSBS gene. N deprivation led to a marked redirection of metabolism: the primary carbon source, acetate, was no longer converted to cell building blocks by the glyoxylate cycle and gluconeogenesis but funneled directly into fatty acid biosynthesis. Additional fatty acids may be produced by membrane remodeling, a process that is suggested by the changes observed in transcript abundance of putative lipase genes. Inferences on metabolism based on transcriptional analysis are indirect, but biochemical experiments supported some of these deductions. The data provided here represent a rich source for the exploration of the mechanism of oil accumulation in microalgae.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Nitrógeno/metabolismo , ARN Mensajero/genética , Secuencia de Bases , Northern Blotting , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/fisiología , Cartilla de ADN , Cromatografía de Gases y Espectrometría de Masas , Expresión Génica , Metabolismo de los Lípidos , Fotosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The development of chloroplasts in Arabidopsis thaliana requires extensive lipid trafficking between the endoplasmic reticulum (ER) and the plastid. The biosynthetic enzymes for the final steps of chloroplast lipid assembly are associated with the plastid envelope membranes. For example, during biosynthesis of the galactoglycerolipids predominant in photosynthetic membranes, galactosyltransferases associated with these membranes transfer galactosyl residues from UDP-Gal to diacylglycerol. In Arabidopsis, diacylglycerol can be derived from the ER or the plastid. Here, we describe a mutant of Arabidopsis, trigalactosyldiacylglycerol4 (tgd4), in which ER-derived diacylglycerol is not available for galactoglycerolipid biosynthesis. This mutant accumulates diagnostic oligogalactoglycerolipids, hence its name, and triacylglycerol in its tissues. The TGD4 gene encodes a protein that appears to be associated with the ER membranes. Mutant ER microsomes show a decreased transfer of lipids to isolated plastids consistent with in vivo labeling data, indicating a disruption of ER-to-plastid lipid transfer. The complex lipid phenotype of the mutant is similar to that of the tgd1,2,3 mutants disrupted in components of a lipid transporter of the inner plastid envelope membrane. However, unlike the TGD1,2,3 complex, which is proposed to transfer phosphatidic acid through the inner envelope membrane, TGD4 appears to be part of the machinery mediating lipid transfer between the ER and the outer plastid envelope membrane. The extent of direct ER-to-plastid envelope contact sites is not altered in the tgd4 mutant. However, this does not preclude a possible function of TGD4 in those contact sites as a conduit for lipid transfer between the ER and the plastid.