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1.
Anal Chim Acta ; 1166: 338545, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34023002

RESUMO

Single nucleotide variants (SNVs) have emerged as increasingly important biomarkers, particularly in the diagnosis and prognosis of cancers. However, most SNVs are rarely detected in blood samples from cancer patients as they are surrounded by abundant concomitant wild-type nucleic acids. Herein, we design a system that features a combination of competitive DNA probe system (CDPS) and duplex-specific nuclease (DSN) that we referred to as CAD. A theoretical model was established for the CAD system based on reaction networks. Guided by the theoretical model, we found that a minor loss in sensitivity significantly improved the specificity of the system, thus creating a theoretical discrimination factor (DF) > 100 for most conditions. This non-equivalent tradeoff between sensitivity and specificity provides a new concept for the analysis of rare DNA-sequence variants. As a demonstration of practicality, we applied as-proposed CAD system to identify low variant allele frequency (VAF) in a synthetic template (0.1% VAF) and human genomic DNA (1% VAF). This work promises complete guidance for the design of enzyme-based nucleic acid analysis.


Assuntos
DNA , Ácidos Nucleicos , DNA/genética , Sondas de DNA/genética , Endonucleases , Humanos , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único
2.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33801827

RESUMO

Here, we summarize a line of remarkably simple, theoretical research to better understand the chemical logic by which life's standard alphabet of 20 genetically encoded amino acids evolved. The connection to the theme of this Special Issue, "Protein Structure Analysis and Prediction with Statistical Scoring Functions", emerges from the ways in which current bioinformatics currently lacks empirical science when it comes to xenoproteins composed largely or entirely of amino acids from beyond the standard genetic code. Our intent is to present new perspectives on existing data from two different frontiers in order to suggest fresh ways in which their findings complement one another. These frontiers are origins/astrobiology research into the emergence of the standard amino acid alphabet, and empirical xenoprotein synthesis.


Assuntos
Aminoácidos/genética , Evolução Molecular , Código Genético/genética , Biossíntese de Proteínas , Proteínas/genética , Algoritmos , Aminoácidos/química , Biologia Computacional/métodos , DNA/química , DNA/genética , Estrutura Molecular , Nucleotídeos/química , Nucleotídeos/genética , Proteínas/química
3.
BMC Bioinformatics ; 22(1): 181, 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33832433

RESUMO

BACKGROUND: The widespread use of next-generation sequencing has identified an important role for somatic mosaicism in many diseases. However, detecting low-level mosaic variants from next-generation sequencing data remains challenging. RESULTS: Here, we present a method for Position-Based Variant Identification (PBVI) that uses empirically-derived distributions of alternate nucleotides from a control dataset. We modeled this approach on 11 segmental overgrowth genes. We show that this method improves detection of single nucleotide mosaic variants of 0.01-0.05 variant allele fraction compared to other low-level variant callers. At depths of 600 × and 1200 ×, we observed > 85% and > 95% sensitivity, respectively. In a cohort of 26 individuals with somatic overgrowth disorders PBVI showed improved signal to noise, identifying pathogenic variants in 17 individuals. CONCLUSION: PBVI can facilitate identification of low-level mosaic variants thus increasing the utility of next-generation sequencing data for research and diagnostic purposes.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Nucleotídeos , Alelos , Estudos de Coortes , Humanos , Nucleotídeos/genética , Software
5.
Med Sci Monit ; 27: e930591, 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33723203

RESUMO

BACKGROUND Cytochrome P450 (CYP) genes are necessary for the production or metabolism of fetal sex hormones during pregnancy. The second-to-fourth digit ratio (2D: 4D) is formed in the early stage of human fetal development and considered an indicator reflecting prenatal sex steroids levels. We explored the association between 2D: 4D and single-nucleotide polymorphisms (SNPs) of CYP. MATERIAL AND METHODS Correlation analysis between 2D: 4D and 8 SNPs, rs2687133 (CPY3A7), rs7173655 (CYP11A1), rs1004467, rs17115149, and rs2486758 (CYP17A1), and rs4646, rs2255192, rs4275794 (CYP19A1), was performed using data from 426 female and 412 male Chinese university students. SNP genotyping was conducted using PCR. Digit lengths were photographed and measured by image processing software. RESULTS rs2486758 (CYP17A1) correlated with left hand 2D: 4D in men (P=0.026), and rs1004467 (CYP17A1) correlated with right hand 2D: 4D in men (P=0.008) and the whole population (P=0.032). In men, allele G rs1004467 decreased right hand 2D: 4D, while allele C of rs2486758 increased left hand 2D: 4D. In women, left hand 2D: 4D was higher in genotypes with allele A of SNP rs4646 (CYP19A1) under the dominant genetic model; female DR-L was higher in genotypes with allele T of rs17115149 (CYP11A1). SNPs rs2687133 (CYP3A7) and rs1004467 (CYP17A1) were significantly correlated with right hand 2D: 4D (P=0.0107). CONCLUSIONS SNPs rs1004467 and rs2486758 of CYP17A1 are significant in the relationship between 2D: 4D and CYP gene polymorphisms under different conditions. SNP interactions between CYP genes probably impact 2D: 4D. The correlation between 2D: 4D and some sex hormone-related diseases may be due to the effect of CYP variants on the 2 phenotypes.


Assuntos
Sistema Enzimático do Citocromo P-450/genética , Dedos/anatomia & histologia , Alelos , Aromatase/genética , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , China , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Citocromo P-450 CYP3A/genética , Feminino , Frequência do Gene/genética , Estudos de Associação Genética/métodos , Genótipo , Humanos , Masculino , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único/genética , Esteroide 17-alfa-Hidroxilase/genética , Estudantes , Universidades , Adulto Jovem
6.
Mol Genet Genomics ; 296(3): 705-717, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33772345

RESUMO

Cytoplasmic male sterility (CMS) observed in many plants leads defect in the production of functional pollen, while the expression of CMS is suppressed by a fertility restorer gene in the nuclear genome. Ogura CMS of radish is induced by a mitochondrial orf138, and a fertility restorer gene, Rfo, encodes a P-type PPR protein, ORF687, acting at the translational level. But, the exact function of ORF687 is still unclear. We found a Japanese variety showing male sterility even in the presence of Rfo. We examined the pollen fertility, Rfo expression, and orf138 mRNA in progenies of this variety. The progeny with Type H orf138 and Rfo showed male sterility when their orf138 mRNA was unprocessed within the coding region. By contrast, all progeny with Type A orf138 were fertile though orf138 mRNA remained unprocessed in the coding region, demonstrating that ORF687 functions on Type A but not on Type H. In silico analysis suggested a specific binding site of ORF687 in the coding region, not the 5' untranslated region estimated previously, of Type A. A single nucleotide substitution in the putative binding site diminishes affinity of ORF687 in Type H and is most likely the cause of the ineffectiveness of ORF687. Furthermore, fertility restoration by RNA processing at a novel site in some progeny plants indicated a new and the third fertility restorer gene, Rfs, for orf138. This study clarified that direct ORF687 binding to the coding region of orf138 is essential for fertility restoration by Rfo.


Assuntos
Proteínas de Arabidopsis/genética , Fertilidade/genética , Genes de Plantas/genética , Nucleotídeos/genética , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Quinases/genética , Raphanus/genética , Regiões 5' não Traduzidas/genética , Aminoácidos/genética , Sequência de Bases , Citoplasma/genética , Regulação da Expressão Gênica de Plantas/genética , Mitocôndrias/genética , Infertilidade das Plantas/genética , Proteínas de Plantas/genética , Pólen/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética
7.
Nat Commun ; 12(1): 874, 2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33558533

RESUMO

Base-pairing interactions mediate many intermolecular target recognition events. Even a single base-pair mismatch can cause a substantial difference in activity but how such changes influence the target search kinetics in vivo is unknown. Here, we use high-throughput sequencing and quantitative super-resolution imaging to probe the mutants of bacterial small RNA, SgrS, and their regulation of ptsG mRNA target. Mutations that disrupt binding of a chaperone protein, Hfq, and are distal to the mRNA annealing region still decrease the rate of target association, kon, and increase the dissociation rate, koff, showing that Hfq directly facilitates sRNA-mRNA annealing in vivo. Single base-pair mismatches in the annealing region reduce kon by 24-31% and increase koff by 14-25%, extending the time it takes to find and destroy the target by about a third. The effects of disrupting contiguous base-pairing are much more modest than that expected from thermodynamics, suggesting that Hfq buffers base-pair disruptions.


Assuntos
Pareamento de Bases/genética , Estabilidade de RNA , RNA Bacteriano/genética , Sequência de Bases , Escherichia coli/genética , Dosagem de Genes , Genes Reporter , Imageamento Tridimensional , Cinética , Mutação/genética , Nucleotídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo
8.
Life Sci ; 271: 119127, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33515561

RESUMO

Gene mutations play important roles in tumour development. In this study, we identified a functional histone H2B mutation H2BL-T11C, causing an amino acid variation from Leu to Pro (L3P, H2BL-L3P). Cells overexpressing H2BL-L3P showed stronger proliferation, colony formation, tumourigenic abilities, and a different cell cycle distribution. Meanwhile, the c-Myc expression was elevated as evident by RNA-seq. We further revealed that an H2BK5ac-H2BK120ubi crosstalk which regulates gene transcription. Moreover, EdU staining demonstrated an important role of c-Myc in accelerating cell cycle progression through the G1/S checkpoint, while treatment with 10058-F4, an inhibitor of the c-Myc/MAX interaction, alleviated the abnormal cell proliferation and cell cycle distribution in vitro and partially inhibited tumour growth in vivo. The mutation of amino acid L3P is associated with tumour progression, suggesting patients carrying this SNP may have higher risk of tumour development.


Assuntos
Proliferação de Células/fisiologia , Variação Genética/genética , Histonas/genética , Neoplasias/genética , Proteínas Proto-Oncogênicas c-myc/genética , Regulação para Cima/fisiologia , Animais , Linhagem Celular Tumoral , Células HEK293 , Histonas/metabolismo , Humanos , Lentivirus , Leucina/genética , Camundongos , Camundongos Nus , Mutação/genética , Neoplasias/metabolismo , Neoplasias/patologia , Nucleotídeos/genética , Prolina/genética , Proteínas Proto-Oncogênicas c-myc/biossíntese , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
9.
Nat Commun ; 12(1): 611, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504776

RESUMO

Genome sequences have been determined for many model organisms; however, repetitive regions such as centromeres, telomeres, and subtelomeres have not yet been sequenced completely. Here, we report the complete sequences of subtelomeric homologous (SH) regions of the fission yeast Schizosaccharomyces pombe. We overcame technical difficulties to obtain subtelomeric repetitive sequences by constructing strains that possess single SH regions of a standard laboratory strain. In addition, some natural isolates of S. pombe were analyzed using previous sequencing data. Whole sequences of SH regions revealed that each SH region consists of two distinct parts with mosaics of multiple common segments or blocks showing high variation among subtelomeres and strains. Subtelomere regions show relatively high frequency of nucleotide variations among strains compared with the other chromosomal regions. Furthermore, we identified subtelomeric RecQ-type helicase genes, tlh3 and tlh4, which add to the already known tlh1 and tlh2, and found that the tlh1-4 genes show high sequence variation with missense mutations, insertions, and deletions but no severe effects on their RNA expression. Our results indicate that SH sequences are highly polymorphic and hot spots for genome variation. These features of subtelomeres may have contributed to genome diversity and, conversely, various diseases.


Assuntos
Variação Genética , Genoma Fúngico , Schizosaccharomyces/genética , Telômero/genética , Sequência de Bases , Mutação INDEL/genética , Mosaicismo , Família Multigênica , Nucleotídeos/genética , Filogenia , RNA Fúngico/genética , RecQ Helicases/genética , Schizosaccharomyces/isolamento & purificação
10.
Nat Protoc ; 16(2): 1089-1128, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462442

RESUMO

Genome editing has transformed the life sciences and has exciting prospects for use in treating genetic diseases. Our laboratory developed base editing to enable precise and efficient genome editing while minimizing undesired byproducts and toxicity associated with double-stranded DNA breaks. Adenine and cytosine base editors mediate targeted A•T-to-G•C or C•G-to-T•A base pair changes, respectively, which can theoretically address most human disease-associated single-nucleotide polymorphisms. Current base editors can achieve high editing efficiencies-for example, approaching 100% in cultured mammalian cells or 70% in adult mouse neurons in vivo. Since their initial description, a large set of base editor variants have been developed with different on-target and off-target editing characteristics. Here, we describe a protocol for using base editing in cultured mammalian cells. We provide guidelines for choosing target sites, appropriate base editor variants and delivery strategies to best suit a desired application. We further describe standard base-editing experiments in HEK293T cells, along with computational analysis of base-editing outcomes using CRISPResso2. Beginning with target DNA site selection, base-editing experiments in mammalian cells can typically be completed within 1-3 weeks and require only standard molecular biology techniques and readily available plasmid constructs.


Assuntos
Adenina/química , Citosina/química , Edição de Genes/métodos , Animais , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , DNA/genética , Quebras de DNA de Cadeia Dupla , Células HEK293 , Humanos , Nucleotídeos/genética
11.
Arch Virol ; 166(2): 655-658, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33394170

RESUMO

RNA was extracted from 'Hugh Dickson' rose leaves displaying virus-like symptoms in Maryland, USA. Using high-throughput sequencing, we identified a new virus, tentatively named "rose virus R". This virus has a negative-sense, single-stranded RNA genome and exhibits genomic features of a rhabdovirus, including a genome organization of 3'-N-P-P3-M-G-P6-L-5' and a gene junction region consensus sequence 3'-AUUUAUUUUGACUCUA-5'. Rose virus R is phylogenetically related to cytorhabdoviruses, and the nucleotide and amino acid sequences of rose virus R and related cytorhabdoviruses have diverged considerably, suggesting that rose virus R should be classified as a member of a novel species in the genus Cytorhabdovirus.


Assuntos
Doenças das Plantas/virologia , Rosa/virologia , Vírus não Classificados/genética , Sequência de Aminoácidos , Genoma Viral/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nucleotídeos/genética , Filogenia , RNA Viral/genética , Rhabdoviridae/genética , Proteínas Virais/genética , Sequenciamento Completo do Genoma/métodos
12.
Arch Virol ; 166(2): 665-669, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33409550

RESUMO

A putative mycovirus belonging to the proposed family "Fusariviridae" was discovered in Setosphaeria turcica by sequencing a double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named "Setosphaeria turcica fusarivirus 1" (StFV1). StFV1 has a genome comprising 6685 nucleotides. The genome contains three open reading frames (ORF). The largest ORF, ORF1, is preceded by an untranslated region (UTR) of 16 nucleotides and separated from ORF2 by an intergenic region of 63 nucleotides. The smallest ORF, ORF3, overlaps ORF2 by 16 nucleotides and is followed by a 3'-UTR of 82 nucleotides. The protein encoded by ORF1 is 71.8%, 67.4% and 68.1% identical to the RNA-dependent RNA polymerases (RdRps) of Pleospora typhicola fusarivirus 1 (PtFV1), Plasmopara viticola lesion-associated fusarivirus 1 (PvlaFV1), and Plasmopara viticola lesion-associated fusarivirus 3 (PvlaFV3), respectively, but has less than 47% amino acid sequence identity to the RdRps of other fusariviruses. To our knowledge, this is the first fusarivirus discovered in S. turcica and the first virus to be identified in this fungus using conventional cloning methods.


Assuntos
Ascomicetos/virologia , Vírus de RNA/genética , Regiões 3' não Traduzidas/genética , Sequência de Aminoácidos , Genoma Viral/genética , Nucleotídeos/genética , Fases de Leitura Aberta/genética , Filogenia , RNA de Cadeia Dupla/genética , RNA Viral/genética , /genética
13.
Mol Phylogenet Evol ; 154: 106961, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32956799

RESUMO

Heliamphora is a genus of carnivorous pitcher plants endemic to the Guiana Highlands with fragmented distributions. We present a well resolved, time-calibrated, and comprehensive Heliamphora phylogeny estimated using Bayesian inference and maximum likelihood based on nuclear genes (26S, ITS, and PHYC) and secondary calibration. We used stochastic mapping to infer ancestral states of morphological characters and ecological traits. Our ancestral state estimations revealed that the pitcher drainage structures characteristic of the genus transformed from a hole to a slit in single clade, while other features (scape pubescence and hammock-like growth) have been gained and lost multiple times. Habitat was similarly labile in Heliamphora, with multiple transitions from the ancestral highland habitats into the lowlands. Using a Mantel test, we found closely related species tend to be geographically closely distributed. Placing our phylogeny in a historical context, major clades likely emerged through both vicariance and dispersal during the Miocene with more recent diversification driven by vertical displacement during the Pleistocene glacial-interglacial thermal oscillations. Despite the dynamic climatic history experienced by Heliamphora, the temperature changes brought by global warming pose a significant threat, particularly for those species at the highest elevations.


Assuntos
Filogenia , Filogeografia , Sarraceniaceae/classificação , Áreas Alagadas , Teorema de Bayes , Funções Verossimilhança , Modelos Biológicos , Nucleotídeos/genética , Fenótipo , América do Sul
14.
Mol Phylogenet Evol ; 154: 106966, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971285

RESUMO

Although numerous studies have demonstrated the theoretical and empirical importance of treating gaps as insertion/deletion (indel) events in phylogenetic analyses, the standard approach to maximum likelihood (ML) analysis employed in the vast majority of empirical studies codes gaps as nucleotides of unknown identity ("missing data"). Therefore, it is imperative to understand the empirical consequences of different numbers and distributions of gaps treated as missing data. We evaluated the effects of variation in the number and distribution of gaps (i.e., no base, coded as IUPAC "." or "-") treated as missing data (i.e., any base, coded as "?" or IUPAC "N") in standard ML analysis. We obtained alignments with variable numbers and arrangements of gaps by aligning seven diverse empirical datasets under different gap opening costs using MAFFT. We selected the optimal substitution model for each alignment using the corrected Akaike Information Criterion in jModelTest2 and searched for optimal trees using GARLI. We also employed a Monte Carlo approach to randomly replace nucleotides with gaps (treated as missing data) in an empirical dataset to understand more precisely the effects of varying their number and distribution. To compare alignments, we developed four new indices and used several existing measures to quantify the number and distribution of gaps in all alignments. Our most important finding is that ML scores correlate negatively with gap opening costs and the amount of missing data. However, this negative relationship is not due to the increase in missing data per se-which increases ML scores-but instead to the effect of gaps on nucleotide homology. These variables also cause significant but largely unpredictable effects on tree topology.


Assuntos
Filogenia , Bases de Dados Genéticas , Funções Verossimilhança , Método de Monte Carlo , Nucleotídeos/genética , Padrões de Referência , Alinhamento de Sequência
15.
Mol Phylogenet Evol ; 156: 107045, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33352317

RESUMO

Ladybirds (family Coccinellidae) are one of the most diverse groups of beetles and globally comprise over 6000 species. Despite their scientific and economic significance, the taxonomy of Coccinellidae remains unstable, and we still know little about their evolutionary history. By using a small number of genes, previous phylogenetic analyses have not reliably resolved the relationships among major ladybird lineages. In this study, we sequenced 94 nuclear protein-coding genes for 214 species of Coccinellidae and 14 outgroups, covering 90 genera and 35 tribes. We found that nucleotide compositional heterogeneity is present among ladybird tribes so that phylogenetic inference at the amino acid level is more reliable than at the DNA level. Based on the maximum likelihood analyses of the amino acid dataset, we recognize three subfamilies in Coccinellidae: Microweiseinae, Monocoryninae stat. nov., and Coccinellinae. The subfamily relationships are strongly supported as (Microweiseinae, (Monocoryninae stat. nov., Coccinellinae)). The tribes of ladybirds are mostly monophyletic, except Ortaliini, Sticholotidini, Scymnini, and Coccidulini. The phylogenetic relationships among tribes of Coccinellinae are still not well resolved, with many nodes weakly supported. Our divergence time analysis suggests that the crown group of extant lady beetles arose in the Early Cretaceous ~ 143 million years ago (Mya) and experienced a rapid diversification during the Late Cretaceous (120-70 Mya). We hypothesize that the boom of angiosperms in the Late Cretaceous promoted the diversification of herbivorous Sternorrhyncha insects, especially aphids, which in turn drove the rapid radiation of predatory lady beetles. In summary, our work provides a comprehensive time-calibrated phylogeny of Coccinellidae that provides a sound framework for revising their classification and understanding the origin of their biodiversity.


Assuntos
Besouros/classificação , Besouros/genética , Genes de Insetos , Filogenia , Aminoácidos/genética , Animais , Composição de Bases/genética , Códon/genética , Nucleotídeos/genética , Fatores de Tempo
16.
Gene ; 766: 145096, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-32919006

RESUMO

The phylogenetic analysis based on sequence similarity targeted to real biological taxa is one of the major challenging tasks. In this paper, we propose a novel alignment-free method, CoFASA (Codon Feature based Amino acid Sequence Analyser), for similarity analysis of nucleotide sequences. At first, we assign numerical weights to the four nucleotides. We then calculate a score of each codon based on the numerical value of the constituent nucleotides, termed as degree of codons. Accordingly, we obtain the degree of each amino acid based on the degree of codons targeted towards a specific amino acid. Utilizing the degree of twenty amino acids and their relative abundance within a given sequence, we generate 20-dimensional features for every coding DNA sequence or protein sequence. We use the features for performing phylogenetic analysis of the set of candidate sequences. We use multiple protein sequences derived from Beta-globin (BG), NADH dehydrogenase subunit 5 (ND5), Transferrins (TFs), Xylanases, low identity (<40%) and high identity (⩾40%) protein sequences (encompassing 533 and 1064 protein families) for experimental assessments. We compare our results with sixteen (16) well-known methods, including both alignment-based and alignment-free methods. Various assessment indices are used, such as the Pearson correlation coefficient, RF (Robinson-Foulds) distance and ROC score for performance analysis. While comparing the performance of CoFASA with alignment-based methods (ClustalW, ClustalΩ, MAFFT, and MUSCLE), it shows very similar results. Further, CoFASA shows better performance in comparison to well-known alignment-free methods, including LZW-Kernal, jD2Stat, FFP, spaced, and AFKS-D2s in predicting taxonomic relationship among candidate taxa. Overall, we observe that the features derived by CoFASA are very much useful in isolating the sequences according to their taxonomic labels. While our method is cost-effective, at the same time, produces consistent and satisfactory outcomes.


Assuntos
Sequência de Aminoácidos/genética , Aminoácidos/genética , Códon/genética , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Algoritmos , Animais , Humanos , Nucleotídeos/genética , Filogenia , Proteínas/genética
17.
Biochem Biophys Res Commun ; 533(4): 1470-1476, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33333712

RESUMO

Exosc2 is one of the components of the exosome complex involved in RNA 3' end processing and degradation of various RNAs. Recently, EXOSC2 mutation has been reported in German families presenting short stature, hearing loss, retinitis pigmentosa, and premature aging. However, the in vivo function of EXOSC2 has been elusive. Herein, we generated Exosc2 knockout (exosc2-/-) zebrafish that showed larval lethality 13 days post fertilization, with microcephaly, loss of spinal motor neurons, myelin deficiency, and retinitis pigmentosa. Mechanistically, Exosc2 deficiency caused impaired mRNA turnover, resulting in a nucleotide pool imbalance. Rapamycin, which modulated mRNA turnover by inhibiting the mTOR pathway, improved nucleotide pool imbalance in exosc2-/- zebrafish, resulting in prolonged survival and partial rescue of neuronal defects. Taken together, our findings offer new insights into the disease pathogenesis caused by Exosc2 deficiency, and might help explain fundamental molecular mechanisms in neuronal diseases, such as Alzheimer's disease, amyotrophic lateral sclerosis, and spinal muscular atrophy.


Assuntos
Nucleotídeos/metabolismo , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas , Embrião não Mamífero/anormalidades , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Larva/genética , Larva/fisiologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Proteína Básica da Mielina/genética , Nucleotídeos/genética , Sirolimo/farmacologia , Peixe-Zebra/embriologia
18.
PLoS Negl Trop Dis ; 14(9): e0008018, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991594

RESUMO

By combining a reference-independent SNP analysis and average nucleotide identity (ANI) with affinity propagation clustering (APC), we developed a significantly improved methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella (F.) tularensis. Molecular epidemiology of F. tularensis is currently assessed mostly based on laboratory methods and molecular analysis. The high evolutionary stability and the clonal nature makes Francisella ideal for subtyping with single nucleotide polymorphisms (SNPs). Sequencing and real-time PCR can be used to validate the SNP analysis. We investigate whole-genome sequences of 155 F. tularensis subsp. holarctica isolates. Phylogenetic testing was based on SNPs and average nucleotide identity (ANI) as reference independent, alignment-free methods taking small-scale and large-scale differences within the genomes into account. Especially the whole genome SNP analysis with kSNP3.0 allowed deciphering quite subtle signals of systematic differences in molecular variation. Affinity propagation clustering (APC) resulted in three clusters showing the known clades B.4, B.6, and B.12. These data correlated with the results of real-time PCR assays targeting canSNPs loci. Additionally, we detected two subtle sub-clusters. SplitsTree was used with standard-setting using the aligned SNPs from Parsnps. Together APC, HierBAPS, and SplitsTree enabled us to generate hypotheses about epidemiologic relationships between bacterial clusters and describing the distribution of isolates. Our data indicate that the choice of the typing technique can increase our understanding of the pathogenesis and transmission of diseases with the eventual for prevention. This is opening perspectives to be applied to other bacterial species. The data provide evidence that Germany might be the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade. Described methods allow generating a new, more detailed perspective for F. tularensis subsp. holarctica phylogeny. These results may encourage to determine phylogenetic relationships and clustering of other bacteria the same way.


Assuntos
Francisella tularensis/classificação , Francisella tularensis/genética , Polimorfismo de Nucleotídeo Único , Genoma Bacteriano , Epidemiologia Molecular , Família Multigênica , Nucleotídeos/genética , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Sequenciamento Completo do Genoma
20.
Proc Natl Acad Sci U S A ; 117(34): 20689-20695, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32788345

RESUMO

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them.


Assuntos
Sequência de Bases/genética , RNA/química , RNA/genética , Sítios de Ligação , DNA/química , Dano ao DNA/genética , DNA Glicosilases/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Desoxirribonuclease I/metabolismo , Humanos , Nucleotídeos/genética , Estruturas R-Loop/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Leveduras/genética
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