1,720,971 research outputs found
Analysis of the architecture and function of the nuclear DNA replication apparatus in Trypanosoma brucei
Get PDFDNA replication is central to the propagation of life and initiates by the designation of genome sequences as origins, where synthesis of a copy of the genetic material begins once per cell division. Despite considerable progress in understanding mitochondrial (kinetoplast) DNA replication in kinetoplastid parasites, little is known about nuclear DNA replication. The mechanism and machinery of DNA replication initiation is well-conserved among characterised eukaryotes. The six protein origin recognition complex (ORC, Orc1-Orc6), Cdc6, and Cdt1 are recruited sequentially to DNA, and once bound, they load the replicative helicase (MCM, a heterohexamer; subunits Mcm2-7) to form a pre-replicative complex (pre-RC) at potential origins of replication. The largest subunit of ORC, Orc1, is related in sequence to Cdc6, indicative of derivation from a common ancestor. Such an ancestral molecule appears still to function in archaea. These prokaryotes lack Cdc6 and possess a protein named Orc1/Cdc6, which appears to provide all ORC functions, since orthologues of Orcs2-6 are absent. In addition to this, archaeal orthologues of Cdt1 have not been clearly described, though potentially related factor, named WhiP (winged helix initiator protein), has been found. Comparative genome analysis of Trypanosoma brucei and related trypanosomatids (Leishmania major and Trypanosoma cruzi) revealed, remarkably, only a single ORC protein that is equally related to eukaryotic Orc1 and Cdc6 (named here TbORC1/CDC6). In addition, no clear homologue of Cdt1 was found. These observations have been interpreted as suggesting that origin designation in trypanosomatids, although eukaryotic, may be archaeal-like, raising numerous mechanistic and evolutionary questions.
To test this hypothesis, and to dissect the process of nuclear DNA replication, a number of experiments are described in this thesis. We used RNA interference (RNAi) to demonstrate that knockdown of TbORC1/CDC6 in procyclic form (PCF) T. brucei cells inhibits nuclear DNA synthesis, as revealed by cell cycle analysis and a BrdU incorporation assay. Immunofluorescence and GFP-tagging showed that in procyclic form (PCF) cells TbORC1/CDC6 is a nuclear protein. In PCF cells, based on the evidence gathered, we confirm that TbORC1/CDC6 acts in nuclear DNA synthesis. In contrast, RNAi knockdown of TbORC1/CDC6 in bloodstream form (BSF) T. brucei cells resulted in the rapid accumulation of cells with more than two nuclei and two kinetoplasts, indicating a deregulation of the cell cycle, which is then followed by cell rapid cell death. This RNAi result provides greater evidence that TbORC1/CDC6 provides an essential function in the parasite, since RNAi depletion of TbORC1/CDC6 in PCF cells has a less pronounced effect on growth. Nevertheless, attempts to generate TbORC1/CDC6 null mutants failed in PCF cells, consistent with an essential role in this life cycle stage also.
To study the molecular interactors of TbORC1/CDC6, we performed immunoprecipitation analyses. From this, we have identified one protein (gene ID, Tb927.10.13380) that acts as a component of the T. brucei pre-replicative machinery, and suggest that this is a previously unidentified orthologue of Orc4. We also indentified a further protein (gene ID, Tb927.10.7980) that may also act in T. brucei DNA replication, but whose identity and function are unclear. TbORC1/CDC6 appears not to interact directly with the TbMCM helicase (for which orthologues of all subunits can be identified), consistent with previous observations from a number of eukaryotic organisms, and contrary to reports in some archaeal species. MCM subunits in T. brucei form at least one subcomplex (TbMCM2/4/6/7) homologous to that previously observed for human, yeast, Drosophila, Xenopus and mouse MCM proteins. Taken together, these data appears to refute the hypothesis that the DNA replication pre-RC machinery in T. brucei is analogous to archaea. Rather, we propose that TbORC contains at least two components, TbORC1/CDC6 and Tb927.10.13380, more analogous to the eukaryotic model, suggesting that origin designation is not carried out by a single protein.
To identify potential replication origin sequences, we performed chromatin immunoprecipitation with functional, epitope-tagged TbORC1/CDC6 in PCF cells and, using a high-resolution tiling array (NimbleGen) for T. brucei, we have mapped TbORC1/CDC6 binding sites along all the megabase chromosomes in the genome. Analyses of chromosomes 1-10 showed that 278 binding sites are sparsely located within the core of chromosomes, of which 114 loci (40%) co-localise with probable RNA Polymerase II transcription start sites, perhaps consistent with an origin function. In addition, a further 330 binding sites are present as high density clusters in subtelomeric VSG arrays, and 81 binding sites are associated with sub-telomeric elements, perhaps consistent with a non-origin function. Consistent with these results, RNAi knockdown of TbORC1/CDC6 led to derepression of metacyclic Variant Surface Glycoprotein (VSG) genes, suggesting that TbORC1/CDC6 plays a role in the epigenetic silencing at VSG expression sites in PCF T. brucei. Similar analysis of VSG expression in BSF cells, and of BSF VSGs in PCF cells, was less conclusive, perhaps suggesting differential functions of TbORC1/CDC6 in different life cycle stages or at different VSG expression sites. These analyses shed new light on the architecture and potential function of TbORC1/CDC6 in T. brucei nuclear DNA replication in general, as well as a potential association between replication and antigenic variation in T. brucei
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
mSphere of influence: modifying an old method to study RNA-protein interactions
Get PDFCalvin Tiengwe works on posttranscriptional gene regulation and iron homeostasis in the parasitic protozoan Trypanosoma brucei. In this mSphere of Influence article, he reflects on how the paper “Comprehensive identification of RNA-protein interactions in any organism using orthogonal organic phase separation (OOPS)” by Queiroz et al. (Nat Biotechnol 37:169–178, 2019, https://doi.org/10.1038/s41587-018-0001-2) influenced his research by providing a tool to capture RNA-protein complexes on a global scale using acid guanidinium thiocyanate-phenol-chloroform (AGPC), an old method hitherto applied for RNA, DNA, or protein purification
Variations on the Author
Get PDF“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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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