RESUMEN
This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage-microbe interactions with nitrogen-fixing symbiotic microorganisms.
Asunto(s)
Bacteriófagos , Genoma Viral , Filogenia , Sinorhizobium , Microbiología del Suelo , Bacteriófagos/genética , Bacteriófagos/aislamiento & purificación , Bacteriófagos/clasificación , Bacteriófagos/fisiología , Sinorhizobium/genética , Sinorhizobium/virología , Sinorhizobium/fisiología , Sistemas de Lectura AbiertaRESUMEN
Bacillus thuringiensis (Bt) is one of the most common entomopathogenic bacteria used as a biopesticide, and source of endotoxin genes for generating insect-resistant transgenic plants. The mechanisms underpinning an insect's susceptibility or resistance to B. thuringiensis are diverse. The bacterial lifecycle does not end with the death of a host, they continue to exploit the cadaver to reproduce and sporulate. Herein, we studied the progression of B. thuringiensis subsp. galleriae infection in two populations of wax moth larvae (Galleria mellonella) to gain further insight into the "arms race" between B. thuringiensis virulence and insect defences. Two doses of B. thuringiensis subsp. galleriae (spore and crystalline toxin mixtures) were administered orally to compare the responses of susceptible (S) and resistant (R) populations at â¼30% mortality each. To investigate B. thuringiensis-insect antibiosis, we used a combination of in vivo infection trials, bacterial microbiome analysis, and RNAi targeting the antibacterial peptide gloverin. Within 48 h post-inoculation, B. thuringiensis-resistant insects purged the midgut of bacteria, i.e., colony forming unit numbers fell below detectable levels. Second, B. thuringiensis rapidly modulated gene expression to initiate sporulation (linked to quorum sensing) when exposed to resistant insects in contrast to susceptible G. mellonella. We reinforce earlier findings that elevated levels of antimicrobial peptides, specifically gloverin, are found in the midgut of resistant insects, which is an evolutionary strategy to combat B. thuringiensis infection via its main portal of entry. A sub-population of highly virulent B. thuringiensis can survive the enhanced immune defences of resistant G. mellonella by disrupting the midgut microbiome and switching rapidly to a necrotrophic strategy, prior to sporulation in the cadaver.
Asunto(s)
Bacillus thuringiensis , Mariposas Nocturnas , Animales , Bacillus thuringiensis/metabolismo , Mariposas Nocturnas/microbiología , Insectos/microbiología , Larva/microbiología , Sistema Digestivo/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.
Asunto(s)
Emulsiones/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , ADN/genética , Cartilla de ADN/genética , Biblioteca de Genes , Genoma/genética , Humanos , Modelos Teóricos , Técnicas de Amplificación de Ácido Nucleico/métodos , Moldes GenéticosRESUMEN
Ribosomal protein uL15 (RPL27a) carries a specific modification, hydroxylation, at the His39 residue, which neighbors the CCA terminus of the E-site-bound tRNA at the mammalian ribosome. Under hypoxia, the level of hydroxylation of this protein decreases. We transiently transfected HEK293T cells with constructs expressing wild-type uL15 or mutated uL15 (His39Ala) incapable of hydroxylation, and demonstrated that ribosomes containing both proteins are competent in translation. By applying RNA-seq to the total cellular and polysome-associated mRNAs, we identified differentially expressed genes (DEGs) in cells containing exogenous uL15 or its mutant form. Analyzing mRNA features of up- and down-regulated DEGs, we found an increase in the level of more abundant mRNAs and shorter CDSs in cells with uL15 mutant for both translated and total cellular mRNAs. The level of longer and rarer mRNAs, on the contrary, decreased. Our data show how ribosome heterogeneity can change the composition of the translatome and transcriptome, depending on the properties of the translated mRNAs.
Asunto(s)
Biosíntesis de Proteínas , Proteínas Ribosómicas , Humanos , Animales , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Hidroxilación , Células HEK293 , Mutación , Mamíferos/metabolismoRESUMEN
Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario.
Asunto(s)
Arterias Mamarias , Humanos , Vasos Coronarios , Células Endoteliales , Multiómica , ProteómicaRESUMEN
Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.
Asunto(s)
Hidrolasas Diéster Fosfóricas , Topotecan , Sistemas CRISPR-Cas , ADN , Reparación del ADN , ADN-Topoisomerasas de Tipo I/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Esterasas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Topotecan/farmacología , Transcriptoma , Poli(ADP-Ribosa) Polimerasa-1/metabolismoRESUMEN
In eukaryotic ribosomes, the conserved protein uS19, formerly known as S15, extends with its C-terminal tail to the decoding site. The cross-linking of uS19 to the A site codon has been detected using synthetic mRNAs bearing 4-thiouridine (s4U) residues. Here, we showed that the A-site tRNA prevents this cross-linking and that the P site codon does not contact uS19. Next, we focused on determining uS19-mRNA interactions in vivo by applying the photoactivatable-ribonucleoside enhancing cross-linking and immunoprecipitation method to a stable HEK293 cell line producing FLAG-tagged uS19 and grown in a medium containing s4U. We found that when translation was stopped by cycloheximide, uS19 was efficiently cross-linked to mRNA regions with a high frequency of Glu, Lys and, more rarely, Arg codons. The results indicate that the complexes, in which the A site codon is not involved in the formation of the mRNA-tRNA duplex, are present among the cycloheximide-arrested 80S complexes, which implies pausing of elongating ribosomes at the above mRNA regions. Thus, our findings demonstrate that the human ribosomal protein uS19 interacts with mRNAs during translation elongation and highlight the regions of mRNAs where ribosome pausing occurs, bringing new structural and functional insights into eukaryotic translation in vivo.
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ARN Mensajero/química , Proteínas Ribosómicas/química , Ribosomas/química , Codón , Eucariontes/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Tiouridina/químicaRESUMEN
First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.
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Codón Iniciador/genética , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genética , Codón Iniciador/ultraestructura , Escherichia coli/genética , Proteínas Fluorescentes Verdes/genética , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/ultraestructura , Ribosomas/genética , Ribosomas/ultraestructuraRESUMEN
A number of mutations in the RPS20 gene encoding the ribosomal protein uS10 have been found to be associated with a predisposition to hereditary non-polyposis colorectal carcinoma (CRC). We transfected HEK293T cells with constructs carrying the uS10 minigene with mutations identical to those mentioned above and examined the effects of the produced proteins on the cellular transcriptome. We showed that uS10 with mutations p.V50SfsX23 or p.L61EfsX11 cannot be incorporated into 40S ribosomal subunits, while the protein with the missense mutation p.V54L functionally replaces the respective endogenous protein in the 40S subunit assembly and the translation process. The comparison of RNA-seq data obtained from cells producing aberrant forms of uS10 with data for those producing the wild-type protein revealed overlapping sets of upregulated and downregulated differently expressed genes (DEGs) related to several pathways. Among the limited number of upregulated DEGs, there were genes directly associated with the progression of CRC, e.g., PPM1D and PIGN. Our findings indicate that the accumulation of the mutant forms of uS10 triggers a cascade of cellular events, similar to that which is triggered when the cell responds to a large number of erroneous proteins, suggesting that this may increase the risk of cancer.
Asunto(s)
Neoplasias Colorrectales , Proteínas Ribosómicas , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Susceptibilidad a Enfermedades , Células HEK293 , Humanos , Mutación , Proteínas Ribosómicas/genética , TranscriptomaRESUMEN
The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.
Asunto(s)
Metiltransferasas , ARN Mensajero/metabolismo , Células HEK293 , Humanos , Metilación , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN Mensajero/químicaRESUMEN
Flow-seq is a method that combines fluorescently activated cell sorting and next-generation sequencing to deduce a large amount of data about translation efficiency from a single experiment. Here, we constructed a library of fluorescent protein-based reporters preceded by a set of 648 natural 5'-untranslated regions (5'-UTRs) of Escherichia coli genes. Usually, Flow-seq libraries are constructed using uniform-length sequence elements, in contrast to natural situations, where functional elements are of heterogenous lengths. Here, we demonstrated that a 5'-UTR library of variable length could be created and analyzed with Flow-seq. In line with previous Flow-seq experiments with randomized 5'-UTRs, we observed the influence of an RNA secondary structure and Shine-Dalgarno sequences on translation efficiency; however, the variability of these parameters for natural 5'-UTRs in our library was smaller in comparison with randomized libraries. In line with this, we only observed a 30-fold difference in translation efficiency between the best and worst bins sorted with this factor. The results correlated with those obtained with ribosome profiling.
Asunto(s)
Escherichia coli , Ribosomas , Escherichia coli/genética , Escherichia coli/metabolismo , Regiones no Traducidas 5'/genética , Ribosomas/genética , Ribosomas/metabolismo , Biblioteca de Genes , Biosíntesis de ProteínasRESUMEN
Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.
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Bacteriófagos , Pectobacterium , Podoviridae , Bacteriófagos/genética , Genoma Viral , Pectobacterium/genética , Filogenia , Podoviridae/genética , PolisacáridosRESUMEN
The protein eL38 is one of the smallest proteins of the mammalian ribosome, which is a component of its large (60S) subunit. The haploinsufficiency of eL38 in mice leads to the Tail-short mutant phenotype characterized by defects in the development of the axial skeleton caused by the poor translation of mRNA subsets of Hox genes. Using the ribosome profiling assay applied to HEK293 cells knocked down of eL38, we examined the effects of the lack of eL38 in 60S subunits on gene expression at the level of translation. A four-fold decrease in the cell content of eL38 was shown to result in significant changes in the translational efficiencies of 150 genes. Among the genes, whose expression at the level of translation was enhanced, there were mainly those associated with basic metabolic processes; namely, translation, protein folding, chromosome organization, splicing, and others. The set of genes with reduced translation efficiencies contained those that are mostly involved in the processes related to the regulation of transcription, including the activation of Hox genes. Thus, we demonstrated that eL38 insufficiency significantly affects the expression of certain genes at the translational level. Our findings facilitate understanding the possible causes of some anomalies in eL38-deficient animals.
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Regulación de la Expresión Génica/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Expresión Génica/genética , Células HEK293 , Humanos , Biosíntesis de Proteínas , ARN Mensajero/genética , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Ribosomas/metabolismo , Transcriptoma/genéticaRESUMEN
Protein uL5 (formerly called L11) is an integral component of the large (60S) subunit of the human ribosome, and its deficiency in cells leads to the impaired biogenesis of 60S subunits. Using RNA interference, we reduced the level of uL5 in HEK293T cells by three times, which caused an almost proportional decrease in the content of the fraction corresponding to 80S ribosomes, without a noticeable diminution in the level of polysomes. By RNA sequencing of uL5-deficient and control cell samples, which were those of total mRNA and mRNA from the polysome fraction, we identified hundreds of differentially expressed genes (DEGs) at the transcriptome and translatome levels and revealed dozens of genes with altered translational efficiency (GATEs). Transcriptionally up-regulated DEGs were mainly associated with rRNA processing, pre-mRNA splicing, translation and DNA repair, while down-regulated DEGs were genes of membrane proteins; the type of regulation depended on the GC content in the 3' untranslated regions of DEG mRNAs. The belonging of GATEs to up-regulated and down-regulated ones was determined by the coding sequence length of their mRNAs. Our findings suggest that the effects observed in uL5-deficient cells result from an insufficiency of translationally active ribosomes caused by a deficiency of 60S subunits.
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Regulación de la Expresión Génica/genética , Proteínas Ribosómicas/deficiencia , Proteínas Ribosómicas/metabolismo , Expresión Génica/genética , Perfilación de la Expresión Génica/métodos , Células HEK293 , Humanos , Biosíntesis de Proteínas/fisiología , Procesamiento Postranscripcional del ARN , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Transcripción Genética/fisiología , Transcriptoma/genéticaRESUMEN
Pseudomonas phage MD8 is a temperate phage isolated from the freshwater lake Baikal. The organisation of the MD8 genome resembles the genomes of lambdoid bacteriophages. However, MD8 gene and protein sequences have little in common with classified representatives of lambda-like phages. Analysis of phage genomes revealed a group of other Pseudomonas phages related to phage MD8 and the genomic layout of MD8-like phages indicated extensive gene exchange involving even the most conservative proteins and leading to a high degree of genomic mosaicism. Multiple horizontal transfers and mosaicism of the genome of MD8, related phages and other λ-like phages raise questions about the principles of taxonomic classification of the representatives of this voluminous phage group. Comparison and analysis of various bioinformatic approaches applied to λ-like phage genomes demonstrated different efficiency and contradictory results in the estimation of genomic similarity and relatedness. However, we were able to make suggestions for the possible origin of the MD8 genome and the basic principles for the taxonomic classification of lambdoid phages. The group comprising 26 MD8-related phages was proposed to classify as two close genera belonging to a big family of λ-like phages.
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Bacteriófago lambda , Genes Virales , Fagos Pseudomonas , Bacteriófago lambda/clasificación , Bacteriófago lambda/genética , Fagos Pseudomonas/clasificación , Fagos Pseudomonas/genéticaRESUMEN
Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application.
Asunto(s)
Bacteriófagos/fisiología , Pectobacterium carotovorum/metabolismo , Pectobacterium carotovorum/virología , Secuencia de Aminoácidos , Bacteriófagos/ultraestructura , Genoma Viral , Genómica , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Filogenia , Polimerizacion , Polisacáridos Bacterianos/química , Unión Proteica , Proteínas Virales/químicaRESUMEN
Gut microbiota is believed to play a crucial role in modulating obesity in humans, and probiotics affecting gut microbiota can alleviate some of the obesity-related health complications. The study was aimed to investigate changes in the composition of the gut microbiome in obese humans due to short-term (2 weeks) treatment of obese patients with a probiotic preparation containing Bifidobacterium longum. Faecal microbiome diversity was studied using the 16S amplicon sequencing by Illumina MiSeq. Bioinformatic analysis showed distribution across 14 phyla (with Firmicutes and Bacteroidetes dominating), 21 class, 125 genera and 973 OTUs. The probiotic treatment decreased relative abundance of Bacteroidetes (Prevotellaceae and Bacteroidaceae), while increasing that of Actinobacteria (Bifidobacteriaceae and Coriobacteriaceae), and Firmicutes (Negativicutes: Veillonellaceae and Clostridia: Peptostreptococcaceae). The probiotic treatment decreased total blood sugar and increased patients' assessment of their physical and mental health. Thus even the short-term Bifidobacterium-based probiotic treatment brought significant compositional changes in the 16S rRNA gene diversity in faecal bacterial assemblages by increasing beneficial and decreasing pathogenic or opportunistic bacteria; the related shifts in life quality assessment necessitate further research into the causal relationships involved.
RESUMEN
Supernumerary elements of the genome are often called B chromosomes. They usually consist of various autosomal sequences and, because of low selective pressure, are mostly pseudogenized and contain many repeats. There are numerous reports on B chromosomes in mammals, fish, invertebrates, plants, and fungi, but only a few of them have been studied using sequencing techniques. However, reptilian supernumerary chromosomes have been detected only cytogenetically and never sequenced or analyzed at the molecular level. One model squamate species with available genome sequence is Anolis carolinensis. The scope of the present article is to describe the genetic content of A. carolinensis supernumerary chromosomes. In this article, we confirm the presence of B chromosomes in this species by reverse painting and synaptonemal complex analysis. We applied low-pass high-throughput sequencing to analyze flow-sorted B chromosomes. Anole B chromosomes exhibit similar traits to other supernumerary chromosomes from different taxons: they contain two genes related to cell division control (INCENP and SPIRE2), are enriched in specific repeats, and show a high degree of pseudogenization. Therefore, the present study confirms that reptilian B chromosomes resemble supernumerary chromosomes of other taxons.
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Cromosomas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Lagartos/genética , Análisis de Secuencia de ADN/métodos , Animales , División Celular , Proteínas Cromosómicas no Histona/genética , Mapeo Cromosómico , Pintura Cromosómica , Evolución Molecular , Proteínas de Microfilamentos/genética , FilogeniaRESUMEN
BACKGROUND: Gut microbiota has been increasingly acknowledged to shape significantly human health, contributing to various autoimmune diseases, both intestinal and non-intestinal, including multiple sclerosis (MS). Gut microbiota studies in patients with relapsing remitting MS strongly suggested its possible role in immunoregulation; however, the profile and potential of gut microbiota involvement in patients with primary progressive MS (PPMS) patients has received much less attention due to the rarity of this disease form. We compared the composition and structure of faecal bacterial assemblage using Illumina MiSeq sequencing of V3-V4 hypervariable region of 16S rRNA genes amplicons in patients with primary progressive MS and in the healthy controls. RESULTS: Over all samples 12 bacterial phyla were identified, containing 21 classes, 25 orders, 54 families, 174 genera and 1256 operational taxonomic units (OTUs). The Firmicutes phylum was found to be ultimately dominating both in OTUs richness (68% of the total bacterial OTU number) and in abundance (71% of the total number of sequence reads), followed by Bacteroidetes (12 and 16%, resp.) and Actinobacteria (7 and 6%, resp.). Summarily in all samples the number of dominant OTUs, i.e. OTUs with ≥1% relative abundance, was 13, representing much less taxonomic richness (three phyla, three classes, four orders, six families and twelve genera) as compared to the total list of identified OTUs and accounting for 30% of the sequence reads number in the healthy cohort and for 23% in the PPMS cohort. Human faecal bacterial diversity profiles were found to differ between PPMS and healthy cohorts at different taxonomic levels in minor or rare taxa. Marked PPMS-associated increase was found in the relative abundance of two dominant OTUs (Gemmiger sp. and an unclassified Ruminococcaceae). The MS-related differences were also found at the level of minor and rare OTUs (101 OTUs). These changes in OTUs' abundance translated into increased bacterial assemblage diversity in patients. CONCLUSION: The findings are important for constructing a more detailed global picture of the primary progressive MS-associated gut microbiota, contributing to better understanding of the disease pathogenesis.
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Bacterias/clasificación , Microbioma Gastrointestinal , Variación Genética , Esclerosis Múltiple Crónica Progresiva/microbiología , Adulto , Anciano , Estudios de Casos y Controles , ADN Bacteriano/genética , Heces/microbiología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Filogenia , ARN Ribosómico 16S/genética , Federación de Rusia , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
Aptamers are short DNA and RNA fragments which bind their molecular targets with affinity and specificity comparable to those of antibodies. Here, we describe the selection of novel 2'-F-RNA aptamers against total human hemoglobin or its glycated form HbA1c. After SELEX and high-throughput sequencing of the enriched libraries, affinities and specificities of candidate aptamers and their truncated variants were examined by the solid-phase bioluminescent assay. As a result, we identified aptamers specific to both hemoglobins or only glycated HbA1c. The developed 2'-F-RNA aptamers have shown their applicability for detection of total and glycated hemoglobin in one sample.