1,721,368 research outputs found
Primer-independent DNA polymerases and their use for DNA synthesis
No full textThe present invention provides an isolated peptide of SEQ ID NO: 1 needed for primase active as well as new replicative DNA polymerase enzymes, preferably that of SEQ ID NO: 2, comprising said peptide. Thus, these DNA polymerases are endowed with priming activity and do not require externally provided primers for initiating and performing DNA amplification. These polymerases are able to carry out a faithful and processive de novo DNA synthesis of DNA templates in the absence of pre-synthetized primers. Therefore, these enzymes of the invention act both as primases and DNA polymerases. Furthermore, they show translesion synthesis capacity, so that they may be useful not only for whole-genome amplification but also for the amplification of damaged DNAs. The invention further refers to methods for amplifying templates DNAs using these enzymesPeer reviewedConsejo Superior de Investigaciones Científicas (España), Institut PasteurA1 Solicitud de patente con informe sobre el estado de la técnic
Les archées thermoacidophiles du genre Saccharolobus : programmés comme un eucaryote, manipulés par un virus, vieillisant comme une tortue
No full textLe cycle cellulaire est une séquence d'événements où la cellule grandit, réplique son génome et se divise. Chez les eucaryotes, ce cycle est régi par divers points de contrôle, tandis que chez les bactéries, les processus de réplication de l'ADN, de ségrégation du génome et de cytokinèse se produisent simultanément. Les archées thermoacidophiles du genre Saccharolobus suivent un programme de cycle semblable à celui des eucaryotes. Les cellules passent par une phase G1 de croissance pré-réplicative ; la phase S de réplication chromosomique; la phase G2, une deuxième période de croissance, puis les phases M et D, correspondant respectivement à la ségrégation du génome et à la cytokinèse. Certains virus eucaryotes perturbent le cycle cellulaire de l'hôte, conduisant à une prolifération accrue qui, dans les organismes multicellulaires, peut provoquer des maladies comme le cancer. Chez les archées, le Saccharolobus islandicus virus STSV2 peut également bloquer le cycle cellulaire de son hôte en phase S. Les cellules infectées passent alors à une division cellulaire asymétrique, suggérant l'existence d'un schéma de vieillissement/rajeunissement. Cette thèse vise à comprendre les changements durant le cycle cellulaire dans les cellules de Saccharolobus et à analyser l'impact de l'infection virale. Parallèlement, j'ai étudié si les paradigmes du vieillissement biologique, généralement appliqués aux organismes multicellulaires, s'appliquent également à cet archéen unicellulaire. Dans le chapitre 1, nous avons analysé les changements transcriptionnels tout au long du cycle cellulaire. Nous avons synchronisé les cultures de S. islandicus et réalisé une analyse transcriptomique d'échantillons enrichis en cellules en phase M-G1, S ou G2. Les analyses d'expression génique et de réseaux de coexpression génique ont permis d'identifier des traits spécifiques à chaque phase, incluant des voies métaboliques, l'appareil de motilité cellulaire et des systèmes de défense antiviraux. Grâce à le machine learning nous avons défini un ensemble robuste de gènes signatures pour chaque phase du cycle. Ces résultats ont fourni une image raffinée du cycle cellulaire de S. islandicus et montrent qu'il ressemble plus à celui des eucaryotes que présumé. Dans le chapitre 2, nous avons étudié l'impact du virus non lytique P2V sur le cycle cellulaire de son hôte, S. solfataricus, un parent de S. islandicus. L'analyse transcriptomique montre que le virus affecte le métabolisme de l'hôte et la division cellulaire, provoquant un arrêt de la croissance. Le croisement du profil transcriptionnel des cellules infectées par P2V avec les ensembles de gènes spécifiques au cycle cellulaire définis dans le chapitre 1 suggère que P2V redirige le cycle cellulaire vers un état de type M-G1. Comme l'infection par le P2V inhibe la division cellulaire, les changements observés sont cohérents avec une inversion de la progression du cycle vers une phase optimale pour la reproduction virale. Au chapitre 3, nous avons examiné si les cellules de S. islandicus présentaient des signes de vieillissement et de rajeunissement. Une analyse des réseaux de coexpression génique à différentes phases du cycle a révélé des caractéristiques du vieillissement biologique. Nous avons observé un pic de mortalité après la division cellulaire, ce qui pourrait signifier une baisse de la mortalité préreproductrice. Pour concilier ces observations, un modèle de vieillissement et de rajeunissement a été proposé, offrant un aperçu des origines évolutives du vieillissement et un nouveau paradigme pour explorer les racines procaryotes du rajeunissement cellulaire eucaryote. Ces études affinent la compréhension du cycle cellulaire chez Saccharolobus, révélant des changements dans l'expression de fonctions cellulaires dépendantes du cycle cellulaire et la capacité d'un virus à manipuler ce cycle. Elles fournissent également des indices sur le vieillissement de cet organisme unicellulaire.The cell cycle is a sequence of events happening as the cell grows, replicates its genome and divides. The eukaryotic cell cycle is controlled by various checkpoints, while bacteria generally lack strong oversight allowing DNA replication, genome segregation and cytokinesis to occur concurrently. Thermoacidophilic archaea of the genus Saccharolobus follow a defined cell cycle program resembling that of eukaryotes: the cells go through (i) G1 phase of pre-replicative growth; (ii) the chromosome replication S phase; (iii) G2 phase, a second period of cellular growth, (iv) genome segregation M phase, and (v) cell division D phase. Some eukaryotic viruses affect the progression of the host cell cycle, leading to enhanced proliferation that, in multicellular organisms, may cause various pathologies, such as cancer. It has been recently demonstrated that Saccharolobus islandicus virus STSV2 blocks the host cell cycle in the S phase, when the genome replication is most active. Moreover, STSV2-infected cells switch to an asymmetric cell division, suggesting the existence of a pattern of aging/rejuvenation. Thus, the main objective of this PhD thesis was to gain a better understanding of the changes taking place throughout the cell cycle in Saccharolobus cells and analyze how virus infection impacts the cell cycle. In parallel, I investigated if paradigms of biological aging and rejuvenation, typically considered in the framework of multicellular organisms, also apply to Saccharolobus cells. In Chapter 1, we analyzed the transcriptional changes occurring during different cell cycle phases. To this end, we synchronized the cultures of S. islandicus and performed an in-depth transcriptomic analysis of samples enriched in cells undergoing the M-G1, S and G2 phases. Differential gene expression and gene co-expression network analyses allowed us to identify many phase-specific traits, which included many metabolic pathways, the cell motility apparatus and certain antiviral defense systems. Using machine learning, we denoted a robust set of signature genes defining each of the cell cycle phases. These results provide a refined picture of the S. islandicus cell cycle and show that it more closely resembles that of eukaryotes than previously appreciated. In Chapter 2, we studied the impact of the virus P2V on the progression of the cell cycle of its host, S. solfataricus, a close relative of S. islandicus. Transcriptomic analysis of the infection time course showed that P2V significantly affects host metabolism and cell division, leading to cell growth arrest. Moreover, cross-referencing the transcriptional profile of P2V-infected cells against the cell cycle phase specific gene sets defined in Chapter 1 indicated that P2V redirects the cell cycle towards M-G1-like state. As P2V infection leads to inhibition of cell division, the observed changes are consistent with a virus-mediated reversal of cell cycle progression towards a phase most optimal for virus reproduction. In Chapter 3, we explored if S. islandicus cells display patterns of aging and rejuvenation. Analysis of gene co-expression networks during different cell cycle phases revealed features of biological aging in S. islandicus. Notably, at the population level, we observed a peak of mortality shortly after cell division, which may signify pre-reproductive mortality decline. To reconcile these observations, we proposed a model of S. islandicus aging and rejuvenation, offering insights into the evolutionary origins of aging and a new paradigm to explore whether cellular senescence and rejuvenation in eukaryotes may have prokaryotic roots. Collectively, the three studies refine the understanding on the cell cycle in Saccharolobus, revealing changes in cell cycle-dependent expression of diverse cellular functions and the ability of a virus to manipulate the cell cycle. We also provide the first clues into aging in this unicellular organism
Isolation et caractérisation de systèmes viraux-hôtes d'archées
No full textLes virus infectant les archées présentent des morphotypes inhabituels et des génomes extrêmement divers. Leur isolation a permis de développer nos connaissances sur la diversité de la virosphère et demeure une piste de recherche primordiale. Durant ma thèse, j’ai isolé et caractérisé de nouveaux virus d'archées et étudié les interactions virus-hôte dans un système modèle bien établi. Des cultures d'enrichissement et des analyses bioinformatiques nous ont permis de décrire de nouveaux virus de crénarchées hyperthermophiles infectant les espèces du genre Pyrobaculum et ainsi de mieux comprendre la diversité architecturale des virus filamenteux. De plus, un provirus a été identifié chez P. oguniense et l’étude de sa réplication a révélé par microscopie électronique des nanostructures pyramidales à la surface des cellules ressemblant à des structures connues de sortie des virions chez des virus de crénarchées. Deux études de protéomique nous ont fourni un aperçu de la dynamique du protéome de Sulfolobus islandicus : l’analyse du protéome de cellules de S. islandicus non infectées a révélé de nombreuses modifications post-traductionnelles, et l’analyse de la régulation des protéines dans des cellules de S. islandicus infectées par le virus SIRV2 a révélé 136 protéines de l'hôte présentant une régulation temporelle significative. L’analyse structurale de SIRV2 a permis d'étudier la résistance des virus crénarchées à des conditions extrêmes et a révélé pour la première fois que la forme A de l'ADN est biologiquement pertinente. Ces résultats ont contribué au développement des connaissances sur la diversité de la virosphère et sur l'évolution des virus d'archées.Viruses infecting Archaea display unusual morphotypes and highly diverse genomes. Several virus-host systems have emerged enabling the detailed characterization of virus-host interplay in archaea. However, isolation of new archaeal viruses proved to be instrumental for expanding the knowledge on the diversity of the Earth’s virosphere. Therefore, I have focused on two major lines of research: isolation of new archaeal viruses and characterization of the virus-host interactions in a well-established model system. A new Pyrobaculum virus with a unique architecture among DNA viruses was described, expanding our knowledge on the diversity of architectural solutions explored by filamentous viruses. Furthermore, attempts to trigger the replication of a provirus in P. oguninese led to the development of six-fold pyramidal nanostructures on the cell surface, resembling known virion egress structures of archeal viruses. Finally, I focused on the interplay between Sulfolobus islandicus and the rod-shaped virus SIRV2. Two proteomic studies yielded insights into the dynamics and posttranslational modifications (PTMs) of the Sulfolobus proteome. Sulfolobus proteins were found to carry a high degree of PTMs on functionally diverse proteins. The global analysis of the regulation of viral and host proteins in SIRV2-infected S. islandicus cells yielded insights the temporal regulation of host and virus proteins. Structural studies on SIRV2 virion have resulted in the first ever description of A-form DNA being a biologically relevant form of DNA. Together, these results contribute to the knowledge on the diversity of the extant virosphere, and the biology and evolution of archaeal viruses
Virus-host interactions during chronic infections & diversity and evolution of Pleolipoviruses
No full textViruses are the most abundant biological entities in any given environment. The have the ability to regulate microbial abundances through their lytic cycles, and actively participate in horizontal gene exchange. Intriguingly, not all viruses kill their hosts, as some establish chronic and long-term interactions with their hosts that result in particle production without cell lysis. So far, our knowledge on the impact of chronic viral infections on host metabolism, as well as, on the diversity of viruses performing this life cycle in prokaryotic systems remains scarce.
In this work, we isolated and characterized a novel pleomorphic virus from the Pleolipoviridae family - Haloferax pleomorphic virus 1 (HFPV-1) - infecting the archaeon Haloferax volcanii. This virus is able to establish a chronic infection in its host that leads to efficient and constant viral release without significant effects on host fitness nor appearance of resistant cells. Remarkably, infection with HFPV-1 prompted a massive remodeling of host transcriptomic program, while specifically inducing the downregulation of host defense mechanisms. Furthermore, we showed that the outcome of the infection is heavily influenced by the cross talk between HFPV-1 and resident proviruses on the host genome. Additionally, we uncovered
that viral infection inhibited the transition into the morphology of host cells in a temperature dependent manner, further highlighting the ecological impacts of chronic infecting viruses. Notably, HFPV-1 exhibits a uniquely broad host range among pleomorphic viruses, as it is capable to infect efficiently a large diversity of hosts including members from different families to the host of isolation, which could prove crucial in the development of novel genetic systems for so far non-manipulable haloarchaea. Furthermore, we greatly expanded the diversity of pleolipoviruses by producing and surveying metagenomic dataset, reveling that the diversity of
pleolipoviruses is larger than previously thought. Thus, we propose a new organization for the Pleolipoviridae family, while also providing further evidence that they likely reshaped their genomes through recombination with different groups of plasmids or other mobile genetic elements.
Our work demonstrates that pleolipoviruses are far more diverse than previously thought and that they can heavily affect host fitness and ecological role, highlight the importance of understanding these chronic-infecting viruses and their impact on host metabolism and ecology in different environments
Recombination between RNA viruses and plasmids might have played a central role in the origin and evolution of small DNA viruses
No full textInternational audienceThe finding that viruses with RNA and DNA genomes can recombine to produce chimeric entities provides valuable insights into the origin and evolution of viruses. It also substantiates the hypothesis that certain groups of DNA viruses could have emerged from plasmids via acquisition of capsid protein-coding genes from RNA viruses
Evolutionary Genomics of Prokaryotic Viruses
No full textEvolutionary history of biological entities is recorded within their nucleic acid sequences and can (sometimes) be deciphered by thorough genomic analysis. In this study we sought to gain insights into the diversity and evolution of bacterial and archaeal viruses. Our primary interest was pointed towards those virus groups/families for which comprehensive genomic analysis was not previously possible due to the lack of sufficient amount of genomic data. During the course of this work twenty-five putative proviruses integrated into various prokaryotic genomes were identified, enabling us to undertake a comparative genomics approach. This analysis allowed us to test the previously formulated evolutionary hypotheses and also provided valuable information on the molecular mechanisms behind the genome evolution of the studied virus groups.Ei saatavillaei saavutettav
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Networks of evolutionary interactions underlying the polyphyletic origin of ssDNA viruses
No full textThis review comes from a themed issue on Virus evolutionEdited by Valerian V Dolja and Mart KrupovicFor a complete overview see the Issue and the EditorialInternational audienceViruses with single-stranded (ss) DNA genomes infect hosts from all three domains of life and are present in all imaginable environments. Many new ssDNA viruses have been recently isolated, including those infecting algae, fungi, insects and even archaea. In parallel, culture-independent metagenomic approaches have illuminated the tremendous genetic diversity of these viruses, yielding valuable insights into their evolution. Here, I integrate this knowledge to propose a scenario in which certain groups of ssDNA viruses (including Geminiviridae, Circoviridae, Parvoviridae and Microviridae) have originated from plasmids via acquisition of jelly-roll capsid protein genes from ssRNA viruses. This scenario places structurally related viruses with DNA and RNA genomes into an evolutionary continuum and highlights general evolutionary trends in the virosphere
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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