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Ανάπτυξη εργαλείων βιοπληροφορικής για πρόβλεψη της πιθανότητας έναρξης της αντιγραφής του DNA σαν συνάρτηση της γονιδιωματικής περιοχής / Development of bioinformatics tools towards the prediction of DNA replication initiation as a function of the genomic region.Λέγουρας, Ιωάννης 22 November 2011 (has links)
Η χρήση της βιοπληροφορικής σε βιολογικά δεδομένα υψηλής απόδοσης είναι μια πολλά υποσχόμενη προσέγγιση για τη δημιουργία νέα γνώσης. Στην
παρούσα εργασία αναλύεται ένα σύνολο δεδομένων που αφορά στα σημεία έναρξης της αντιγραφής (αφετηρίες) στο ζυμομύκητα Schizosaccharomyces pombe, όπως αναγνωρίστηκαν από πειράματα μικροσυστοιχιών που
κάλυπταν όλο το μήκος του γονιδιώματος του οργανισμού (full genome). Οι αντιγραφή ξεκινάει από μεγάλο αριθμό αφετηριών οι οποίες βρίσκονται
διάσπαρτες σε όλο το γονιδίωμα και μέχρι τώρα οι περισσότερες μελέτες των χαρακτηριστικών των αφετηρίων είχαν πραγματοποιηθεί για περιορισμένο
αριθμό αυτών. Στην εργασία αυτή αναλύονται για πρώτη φορά τα χαρακτηριστικά του συνόλου των αφετηριών του S. pombe με σκοπό να διαπιστωθεί ποια χαρακτηριστικά καθορίζουν πότε μια περιοχή του
γονιδιώματος μπορεί να δράσει ως αφετηρία αντιγραφής. Από την ανάλυση αυτή προκύπτει ότι:
1. Οι αφετηρίες έχουν υψηλότερο μέγιστο περιεχόμενο ΑΤ από άλλες γονιδιωματικές περιοχές.
2. Οι αφετηρίες εντοπίζονται κατά προτίμηση σε μεγάλες διαγονιδιακές περιοχές ανάμεσα σε αποκλίνουσες μεταγραφικές μονάδες.
3. Η ασυμμετρία κατανομής Α και Τ ενδέχεται να αποτελεί δείκτη των αφετηριών.
4. Η απόδοση έχει συσχέτιση με το περιεχόμενο ΑΤ. / Use of Bioinformatics in high-throughput biological data is a promising approach for creation of new knowledge. In this work we analyze a dataset that concerns origins of DNA replication initiation in the yeast Schizosaccharomyces pombe, that were identified through full genome microarray experiments. DNA replication starts from a large number of origins that span the entire genome and until recently most studies of origins of replication have been carried out only for a limited number of them. Here we analyze for the first time the properties of the entire dataset of origins of replication in S. pombe in order to find out which specific properties define which genomic location can function as an origin of replication. From this analysis we found that:
1. Origins of replication have higher maximum AT (adenine-thymine) content than other genomic locations.
2. Origins of replication are found preferentially in large genomic locations between divergent transcriptional units.
3. AT asymmetry might be a marker of origins of replication.
4. The origin of replication firing efficiency is correlated with AT content.
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Rôle de l'interférence à l'ARN et de Mmi1 dans la régulation de la différenciation sexuelle chez le Schizosaccharomyces pompe / Role of RNA interference and Mmi1 in the regulation of sexual differentiation of Schizosaccharomyces pombe.Vavasseur, Aurelia 27 September 2011 (has links)
L'interférence à l'ARN (RNAi) est un mécanisme cellulaire connu pour inhiber l'expression de gènes avec une grande spécificité de séquence. Chez la levure Schizosaccharomyces pombe, ce processus induit des modifications de structure de la chromatine et implique une interaction entre un ARN naissant et un petit ARN associé au complexe du RNAi, RITS (RNA-induced Initiation of Transcriptional gene Silencing). RITS cible les régions répétées et non codantes et joue un rôle essentiel dans l'intégrité de l'hétérochromatine de ces sites génomiques. Une étude a mis en évidence la présence de la sous-unité Argonaute 1 du complexe RITS, ainsi qu'une marque de l'hétérochromatine, la méthylation de la lysine 9 de l'histone H3 (H3K9me), au niveau de la chromatine de deux gènes méiotiques, mei4 et ssm4. Ceci suggérait une nouvelle fonction du RNAi dans la différenciation sexuelle. Au cours de ma thèse, j'ai montré que la protéine de liaison à l'ARN Mmi1 (Meiotic mRNA interception protein 1), permet à RITS de s'associer spécifiquement à la chromatine et à l'ARN messager de ces gènes méiotiques. La protéine Mmi1 orchestre une répression post-transcriptionnelle de gènes méiotiques spécifiques, une activité de « silencing » essentielle au contrôle de la différenciation sexuelle. Nous avons mené une analyse de l'ensemble du transcriptome dans une souche déficiente pour Mmi1, ce qui nous a conduits à l'identification de nouveaux ARNm méiotiques ciblés directement par Mmi1 et le RNAi. Curieusement, la chromatine des gènes méiotiques correspondants ne présente pas systématiquement la marque épigénétique répressive H3K9me, ce qui suggère que le RNAi pourrait réprimer certains gènes codants seulement au niveau post-transcriptionnel. En parallèle, en combinant des techniques de génétique, de biologie moléculaire et de physiologie cellulaire, nous mettons en évidence un probable rôle direct du RNAi dans l'inhibition de la différenciation sexuelle. Nous proposons que le RNAi pourrait coopérer avec Mmi1 pour bloquer de manière efficace une partie du programme transcriptionnel méiotique durant le cycle végétatif. Cette régulation serait essentielle pour l'activation appropriée de ce programme au cours de la progression de la différenciation sexuelle. / RNA interference (RNAi) is a cellular process known for inhibiting gene expression in a sequence-specific manner. In the fission yeast Schizosaccharomyces pombe, this process induces modifications in chromatin structure and is assumed to involve an interaction between nascent transcripts and a small RNA contained in the RNAi complex, RITS (RNA-induced Initiation of Transcriptional gene Silencing). RITS targets repeated and non-coding regions, and is essential for heterochromatin integrity at these genomic sites. In one study, RITS complex subunit Argonaute 1, and a heterochromatin mark, methylation of histone H3 on lysine 9 (H3K9me), were detected on chromatin of two meiotic genes, mei4 and ssm4. This finding suggested a possible new function for RNAi in sexual differentiation. During my PhD studies, I found that a RNA-binding protein, Mmi1 (Meiotic mRNA interception protein 1), enables RITS to specifically associate with the chromatin and messenger RNAs of these meiotic genes. Mmi1 protein triggers a post-transcriptional repression of specific meiotic genes, a silencing activity essential for the control of sexual differentiation. We conducted a genome wide transcriptomic analysis from a mmi1Δ strain, and uncovered additional meiotic mRNAs that are directly targeted by both Mmi1 and RNAi. Intriguingly, chromatin of the corresponding meiotic genes does not necessarily display the repressive epigenetic mark H3K9me, suggesting that RNAi might silence some protein-coding genes only at a post-transcriptional level. In parallel, combining genetic, molecular biology and physiological techniques, we highlighted a potentially direct role for RNAi in the inhibition of sexual differentiation. We propose that RNAi cooperates with Mmi1 to efficiently block expression of the early meiotic transcriptional programme during vegetative growth. This regulation might be essential for the proper timing of activation of this programme during sexual differentiation progression.
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Importance of the Clr2 protein in heterochromatin formation in the fission yeast Schizosaccharomyces pombeSteinhauf, Daniel January 2017 (has links)
Epigenetics is an area of biology that studies heritable changes in gene ex- pression without any change in the DNA sequence. The most studied epige- netic mechanisms are DNA methylation, RNA interference and histone mod- ifications. There are over 130 different modifications that can be attached to histones, and the most commonly studied are methylation, acetylation, phos- phorylation, sumoylation and ubiquitination. The modifications, spread out through the genome, form the histone code, which recruits transcription fac- tors and modifies the accessibility of the DNA, which results in either active or silenced transcription. The silenced form of chromatin is known as heter- ochromatin and is usually found in regions of the chromosome that need to be highly regulated. To study epigenetics, the model organism Schizosac- charomyces pombe is used widely used. S. pombe is a single cell, rod shaped, fission yeast. The simplicity of S. pombe and its similarities to high- er eukaryotes makes it a good model organism for studying epigenetics. We find that, when mutating evolutionary conserved amino acids in the Clr2 protein, which is involved in heterochromatin formation in S. pombe, there is a change in silencing in different heterochromatic regions. When constructs of Clr2 with the BAH domain deleted are overexpressed, there is an increase in silencing in the central core centromere of chromosome II of S. pombe.
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Characterization of Putative Mammalian Adenylyl Cyclase Inhibitors Using the Fission Yeast Schizosaccharomyces pombePacella, Daniel January 2022 (has links)
Thesis advisor: Charles Hoffman / In both mammals and fission yeast, control of cAMP levels is maintained by adenylyl cyclases (ACs), which synthesize cyclic nucleotide, and by cyclic nucleotide phosphodiesterases (PDEs), which are responsible for its degradation. AC activity is regulated by G proteins, which respond to signals from G protein-coupled receptors (GPCRs) that detect extracellular signaling factors such as hormones. cAMP is a second messenger that has several effectors, with protein kinase A (PKA) being a primary target of activation that phosphorylates several downstream targets and results in modulation of pathways such as cell growth and gluconeogenesis. Aberrant cAMP regulation has been linked to several human disease states, such as McCune-Albright Syndrome, which is the result of elevated cAMP levels. Whereas the targeting of PDEs with drugs and selective inhibitors has been very successful, the AC-inhibiting compounds identified to date are unfavorable for clinical use. Inhibitors may not necessarily bind to and inhibit a given AC directly but instead act on a regulatory pathway such as calmodulin signaling. Theoretically, they also may bind to the G protein, interfere with the AC-G protein stimulatory complex, or regulate a factor of AC transcription. Since more than one AC species is expressed in many human cell types, it is difficult to selectively reduce cAMP levels. Therefore, for an AC inhibitor to be favored as a candidate for drug development, it is likely that the compound should directly bind to and inhibit the AC. This thesis describes my studies on a scaffold of 41 structurally related BCAC compounds, called the BCAC51 scaffold, that was identified in a high-throughput screen (HTS) with Schizosaccharomyces pombe strains transformed with GNAS and either mammalian AC4 or AC7. I carried out a series of experiments to examine whether the compounds bind to and inhibit mammalian ACs directly. The most active compounds were further characterized for potency and specificity against a panel of ACs. Several compounds significantly reduced cAMP production, but it could not be determined if the compounds directly or indirectly altered AC activity. I also cloned and constructed strains expressing the human wild-type AC5 gene and the AC5 R418W mutant, which has shown an increased sensitivity to GNAS. cAMP assays on these strains using various BCAC compounds showed that while most compounds had similar effects on both forms of AC5, BCAC62 was significantly more effective on the wild-type enzyme than on the mutant AC5, although the reason for this is unclear. To test whether the compounds could reduce AC activity in the absence of GNAS (basal activity), a flow cytometry study was carried out using a PKA-repressed GFP reporter. Results suggested that BCAC compounds do reduce basal-AC activity and therefore do not act by binding to and inhibiting GNAS, by interfering with the AC-GNAS stimulatory complex, nor by stimulating PDE. Finally, I developed a molecular genetic screen for mutant alleles of an AC gene that confer compound-resistance. One cycle of the screen is near completion, and the screen provides a foundation for future examination of compound-resistant AC candidates. The results presented in this thesis serve as a basis for further research into members of the BCAC51 compound series being putative direct inhibitors of mammalian ACs. / Thesis (BS) — Boston College, 2022. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Scholar of the College. / Discipline: Biology.
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Divergent Evolution of Eukaryotic CC- and A-Adding EnzymesErber, Lieselotte, Franz, Paul, Betat, Heike, Prohaska, Sonja, Mörl, Mario 26 January 2024 (has links)
Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes
or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence
of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria,
the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe.
Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding
enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted
activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their
bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within
a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the
CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely
related enzymes with full activity was able to restore CCA-addition, corroborating the significance of
this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data
demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the
same mechanistic principles but, surprisingly, originate from different evolutionary events.
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Identification of Mutations that Extend the Fission Yeast <i>Schizosaccharomyces pombe</i> Chronological Lifespan by a Novel Parallel Selection ApproachChen, Bo-Ruei 07 July 2011 (has links)
No description available.
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Regulation of Septum Formation by Two Novel Proteins Art1 and Bga1 in Fission Yeast CytokinesisDavidson, Reshma 29 December 2016 (has links)
No description available.
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Papel de la peroxirredoxina Tpxl y del factor de trascripción Pap1 en la respuesta a H2O2 en Schizossaccharomyces pombeVivancos Prellezo, Ana 02 June 2006 (has links)
La vida aeróbica conlleva la formación de especies reactivas derivadas del oxígeno: el radical hidroxilo (OH·), el ión superóxido (O2·-) y el peróxido de hidrógeno (H2O2). En Schizosaccharomyces pombe, dos rutas controlan las respuestas antioxidantes en respuesta a estrés oxidativo por H2O2: la del factor de transcripción Pap1 y la de la MAP quinasa Sty1. En esta tesis doctoral, hemos determinado que la activación de Pap1 se da en respuesta a dosis moderadas, pero no severas, de H2O2. Hemos identificado a la peroxirredoxina Tpx1 como sensor y transmisor de la señal de estrés oxidativo a Pap1. La inactivación temporal de Tpx1, durante estrés oxidativo severo, por oxidación a sulfínico de su cisteína catalítica inhibe la transmisión de señal a Pap1. En dichas condiciones, se activa la ruta de Sty1, que media la inducción de Srx1, cuya función es reducir y, con ello, reactivar a Tpx1. Finalmente, hemos estudiado el papel esencial de Tpx1 en aerobiosis. / Aerobic life involves formation of reactive oxygen species: hydroxyl radical (OH·), superoxide ion (O2·-) and hydrogen peroxide (H2O2). In Schizosaccharomyces pombe, two pathways respond to H2O2 and trigger independent antioxidant-gene responses: the Pap1 and the Sty1 pathways. In this thesis project, we have determined that the activation of the transcription factor Pap1 occurs only at low, but not elevated, H2O2 concentrations. We have identified the peroxiredoxin Tpx1 as a H2O2-sensor and redox activator of Pap1. The temporal inactivation of Tpx1 during severe oxidative stress, by oxidation of its catalytic cysteine to sulfinic acid, inhibits signal transduction to Pap1. During these conditions, the MAP kinase Sty1 is activated and expression of the sulfiredoxin Srx1 is triggered. Srx1 functions to reduce and thus reactivate Tpx1. Finally, we have analysed the essential function of Tpx1 in aerobiosis.
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Promoter-driven splicing regulation in fission yeastMoldón Vara, Alberto 17 October 2008 (has links)
The meiotic cell cycle is modified from the mitotic cell cycle by having a premeiotic S phase which leads to high levels of recombination, two rounds of nuclear division with no intervening DNA synthesis, and a reductional pattern of chromosome segregation. Rem1 is a cyclin that is expressed only during meiosis in the fission yeast Schizosaccharomyces pombe. Cells in which rem1 has been deleted show a decreased intragenic meiotic recombination and a delay at the onset of meiosis I. When ectopically expressed in mitotically growing cells, Rem1 induces a G1 arrest followed by severe mitotic catastrophes. Here we show that rem1 expression is regulated at the level of both transcription and splicing, encoding for two proteins with different function depending on the intron retention. We have determined that the regulation of rem1 splicing is not dependent on any transcribed region of the gene. Furthermore, when the rem1 promoter is fused to other intron-containing genes, the chimeras show a meiotic-specific regulation of splicing, exactly as endogenous rem1. This regulation is dependent on two transcription factors of the forkhead family, Mei4 and Fkh2. While Mei4 induces both transcription and splicing of rem1, Fkh2 is responsible for the intron retention of the transcript during vegetative growth and pre-meiotic S phase. / El ciclo meiótico se diferencia del ciclo mitótico por tener una fase S pre-meiótica caracterizada por altos niveles de recombinación, dos rondas de división nuclear sin síntesis de DNA entre las dos y una segregación cromosómica reduccional. Rem1 es una ciclina que sólo se expresa en meiosis en la levadura de fisión Schizosaccharomyces pombe. Celulas con rem1 deleccionado presentan una tasa de recombinación intragénica disminuida y un retraso en el inicio de meiosis I. Cuando se expresa ectópicamente en células creciendo vegetativamente, Rem1 induce un arresto en G1 seguido de catástrofe mitótica. Este trabajo describe que la expresión de rem1 está regulada a nivel de la trascripción y el procesamiento, codificando para dos proteínas con funciones diferentes dependiendo de la retención intrónica.. Hemos determinado que la regulación del splicing de rem1 no depende de ninguna región transcrita del gen. Además, cuando el promotor se fusiona a otros genes que contienen intrones, las quimeras presentan una regulación específica de meiosis como el rem1 endógeno. Esta regulación depende de dos factores de transcripción de la familia Forkhead, Mei4 y Fkh2. Mientras Mei4 induce la transcripción y el splicing de rem1, Fkh2 es responsable de la retención intrónica del tránscrito durante crecimiento vegetativo y fase S pre-meiótica.
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Vlastnosti DNA vazebných mutant proteinů CSL / Vlastnosti DNA vazebných mutant proteinů CSLTeska, Mikoláš January 2012 (has links)
Notch pathway plays a critical role during the development and life of metazoan organisms. CSL proteins are the component of the Notch pathway that mediates the regulation of target genes. The discovery of CSL-like proteins in yeast raised the question of their function in unicellular organisms which did not utilize the canonical Notch pathway. CSL-homologues in yeast are conserved in parts that are important for DNA binding and for fission yeast proteins it was shown that they bind to CSL recognition elements in vitro. In fission yeast, CSL paralogues Cbf11 and Cbf12 play antagonistic roles in cell adhesion and the coordination of cell and nuclear division. Yeast CSL proteins have long and intrinsically unstructured N- terminal domains compared to metazoan CSL proteins. In this study, we investigated the functional significance of these extended N-termini of CSL proteins by their complete removal. For newly constructed truncated variants of proteins Cbf11 and Cbf12 in Schizosaccharomyces pombe we observed the lack of ability to bind CSL recognition RBP probe. The removal of N-terminal parts of CSL proteins in fission yeast led to the change in their cellular localization. Once strongly preferred nuclear localization changed by the removal of N-terminal domains to cytoplasmic localization with a...
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