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Mata, Monteagudo Juan Ignacio. "Fission yeast cell polarity." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265407.
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Spink, Karen Gillian. "Telomeric proteins in fission yeast." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312057.
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Hansen, Karen. "3' end formation in fission yeast." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389053.
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Beck, Timothy Joseph. "A phenotype ontology for fission yeast." Thesis, University of Sussex, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488618.
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This work examines the suitability of two different ontology approaches for the annotation of Schizosaccharomyces pombe (fission yeast) phenotypes derived from a number of screens of two fission yeast strain libraries- a temperature-sensitive library and an insertional library.
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Woollard, Alison. "Cell cycle control in fission yeast." Thesis, University of London, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318479.
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Abbott, Johanna. "Novel kinetochore factors in fission yeast." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/11825.
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The fission yeast centromere is packaged as transcriptionally silent heterochromatin which serves as a platform for kinetochore assembly. The centromere consists of two distinct domains; the outer repeats and the central core. It has been shown previously that these regions associate with distinct sets of proteins, for example, the fission yeast homologue of CENP-A, Cnp1p, is present at the central core, together with Mis6 and Mis12, whilst the heterochromatin protein Swi6 associates with the outer repeats. Marker genes placed in the centromere are transcriptionally silenced. This feature of the fission yeast centromere was utilised to screen for potential kinetochore components or regulators. Mutants with alleviated silencing at the central core were isolated and seven complementation groups identified; sim1 to 7, for silencing in the middle of the centromere. All the sim mutants display chromosome segregation defects and elevated rates of loss of a non-essential minichromosome. This study describes the ongoing characterisation of sims 1,6 and 7. GFP tagged Sim1 associates with the central core of the centromere suggesting that Sim1 is also a novel kinetochore protein. Our working model is that Sim1 may be required for the assembly of Cnp1p chromatin. The sim6 mutant is unusual as it alleviates silencing at both the central core and outer repeat regions. In the sim7 mutant at the restrictive temperature, Cnp1, a crucial component of the centromere, shows greatly reduced localisation.
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Atkinson, S. R. "The fission yeast non-coding transcriptome." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1457868/.
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Long non-coding RNAs (lncRNAs) are emerging as important regulators of gene expression, although it remains unclear to what extent they contribute overall to the information flow from genotype to phenotype. Using strand-specific RNAsequencing, I identify thousands of novel unstable, or cryptic, lncRNAs in Schizosaccharomyces pombe. The nuclear exosome, the RNAi pathway and the cytoplasmic exonuclease Exo2 represent three key pathways regulating lncRNAs in S. pombe, defining the overlapping classes of CUTs, RUTs and XUTs, respectively. The nuclear exosome and the RNAi pathway act cooperatively to control nuclear lncRNA expression, while the cytoplasmic Exo2 pathway is more distinct. Impairing both the nuclear exosome and the cytoplasmic exonuclease Exo2 is lethal in S. pombe. Importantly, I show that CUTs, RUTs and XUTs are stabilised under physiologically relevant growth conditions, with three key groups emerging: late meiotic RUTs/XUTs, early meiotic CUTs and quiescent CUTs. Late meiotic RUTs/XUTs tend to be antisense to protein-coding genes, and anti-correlate in expression with their sense loci. In contrast, early meiotic and quiescent CUTs tend to be transcribed divergently from protein-coding genes and positively correlate in expression with their mRNA partners. The current study provides an in-depth survey of the lncRNA repertoire of S. pombe, and the pathways that regulate their expression. It seems likely that any regulatory functions mediated by most of these lncRNAs are in cis, nuclear and cotranscriptional. The current study provides a rich and comprehensive resource for future studies of lncRNA function.
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Scheffler, Kathleen. "Microtubule-dependent nuclear congression in fission yeast and a novel factor in cellular morphogenesis of fission yeast." Thesis, Paris 6, 2014. http://www.theses.fr/2014PA066510/document.
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(I) J'ai étudié les mécanismes contrôlant la congression des noyaux pendant la conjugaison de la levure S. pombe. A l'aide d'imagerie à long terme basée sur la microfluidique, j'ai mesuré la durée précise de la congression nucléaire et démontré que deux moteurs moléculaires des MTs, la dynéine et la kinésine-14 Klp2 contribuent à ce processus, dans des voies parallèles. La dynéine s’associe aux SPBs. Son niveau au SPB dépend de la chaine légère intermédiaire Dli1 qui pourrait potentiellement stabiliser le complexe dynéine et est requise pour la congression. Klp2 se localise sur les MTs. La localisation différentielle des deux moteurs suggère des rôles distincts pour tirer les noyaux l'un vers l'autre. Klp2 pourrait induire le glissement de MTs antiparallèles émanant des SPBs, alors que la dynéine localisée au SPB pourrait tirer sur des MTs émanant du SPB opposé.(II) J'ai caractérisé un nouveau facteur morphogénétique, l’AAA+-ATPase Knk1, qui promeut la croissance linéaire chez S. pombe. L’absence de Knk1 provoque la formation d’un coude à proximité des extrémités cellulaires. Ce défaut ne résulte pas de défauts des MTs, qui participent à la linéarité de la croissance. Knk1 se localise aux extrémités de la cellule indépendamment des MTs et des câbles d’actine. Cette localisation requiert son N-terminus et est renforcée quand le domaine ATPase C-terminal lie l’ATP. La concentration de Knk1 aux extrémités est aussi contrôlée par Sla2 et Cdc42, de manière anti-correlée, et indépendamment de l’endocytose. Enfin, Knk1 oscille périodiquement entre les deux extrémités, indépendamment des oscillations de Cdc42, suggérant l'existence d'au moins deux systèmes oscillatoires séparés(I) I studied the molecular mechanisms underlying nuclear congression during fission yeast conjugation. Using microfluidic-based long-term imaging, I defined the precise timing of nuclear congression compared to cell mating and found that two MT molecular motors, dynein and the kinesin-14 Klp2 promote nuclear congression in parallel pathways. Dynein associates with SPBs. Dynein level at SPBs is controlled by the light intermediate chain Dli1 that may promote stabilization of the dynein complex and is essential for dynein-dependent nuclear congression, while dynactin is surprisingly not required for this process. Klp2 localizes along MTs. These differential localization patterns suggest distinct roles for the two motors in pulling the nuclei together: Klp2 may slide anti-parallel MTs emanating from the SPBs, while dynein at the SPB may pull on MTs emanating from the opposite SPB.(II) I characterized a novel morphogenetic factor, the AAA+-ATPase Knk1, supporting linear growth in fission yeast. knk1Δ cells display a kink close to cell tips, a unique shape phenotype that is neither caused by defects in behavior of MTs that promote linear extension. Knk1 localizes to cell tip independently of MTs and actin cables. This localization is mediated by Knk1 N-terminus and enhanced upon ATP binding to Knk1 C-terminal ATPase domain. Knk1 tip levels are enhanced in a sla2 or cdc42, independently of Sla2 role in endocytosis. Finally, Knk1 oscillates between the two cell tips in an anti-correlated periodic manner possibly uncoupled from Cdc42 oscillations suggesting the existence of at least two separated oscillatory systems contributing to fission yeast morphogenesis
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Moldón, Vara Alberto. "Promoter-driven splicing regulation in fission yeast." Doctoral thesis, Universitat Pompeu Fabra, 2008. http://hdl.handle.net/10803/7125.
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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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SAKALAR, Cagri. "Roles of H2A.z in Fission Yeast Chromatin." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2007. http://nbn-resolving.de/urn:nbn:de:swb:14-1195137345841-32085.
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Covalent histone modifications such as methylation, acetylation as well as differential incorporation of histone variants are shown to coincide with different chromatin compartments and mark active or repressed genes. Msc1 is one of the seven JmjC Domain Proteins (JDPs) in Fission Yeast. JDPs are known to function in chromatin and some act as histone demethylases. We found that Msc1 is a member of Swr1 Complex which is known to exchange histone H2A variant H2A.z in nucleosomes. We purified H2A.z as a member of Swr1 Complex and its interaction with Swr1 Complex depends on Swr1. We’ve shown that histone H4 Lysine 20 trimethylation (H4 K20 Me3) is lost in h2A.z and msc1 deletion strains and these strains are sensitive to UV. Deletion strain of h2A.z is sensitive to Camptothecin. Histones H3 and H4 are obtained in Msc1 and H2A.z purifications and we’ve shown that histone H4 from these purifications has low level of Lysine 16 acetylation (H4 K16 Ac). Deletion strains of h2A.z, swr1 and msc1 are shown to be sensitive to TSA, a histone deacetylase (HDAC) inhibitor suggesting that H2A.z cooperates with HDACs. TSA treatment of wild type cells cause an increase in H4 K16 Ac and a decrease in H4 K20 Me3. Gene expression profiles of h2A.z, swr1 and msc1 are significantly similar and upregulated genes in deletion strains localize at chromosome ends (a region of 160 kb for each end). The number of stress or meiotic inducible genes is increased in deletion strains suggesting that H2A.z has a role in regulation of inducible genes. We suggest that H2A.z, in cooperation with HDACs, functions in regulation of chromatin accessibility of inducible promoters.
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Ocampos, Maristela. "A study of arginase in fission yeast." Thesis, University of Cambridge, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388437.
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Heichinger, Christian. "Characterisation of fission yeast DNA replication origins." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1444737/.
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In many eukaryotic organisms the chromosomal origins of DNA replication (ORIs) are not characterised by a clearly defined consensus sequence. In this thesis using the fission yeast, for the first time I have carried out a genome-wide analysis to identify such ORIs during the mitotic and meiotic cell cycles. The data can be summarised as follows: a total of 401 ORIs were identified which were used 29 percent of the time during mitotic S-phase and were spaced every 31 kilobases (kb) on average. The same ORIs were used during pre-meiotic S-phase although with lower efficiency in most chromosomal regions. A further 503 potential ORIs were used less efficiently at eight percent of the time during mitotic S-phase. This totals 904 ORIs which were distributed at an average inter-origin distance of 14 kilobases (kb) throughout 12.5 megabases (Mb) of the three chromosomes of fission yeast. These data support the idea of a continuum of ORI activity. The 401 efficient ORI loci contained A+T-rich regions located between genes, and these intergenic regions were typically larger than average. ORIs were not defined by a strict sequence consensus but the presence of AT-hook binding sequences. When the initiation factors Cdc18 and Cdt1 were over-expressed, regions of DNA containing particularly efficient ORIs with exceptionally large AT-hook binding domains became over-amplified, suggesting that interactions between these factors and efficient ORIs may be important for the mechanism ensuring that an ORI only fires once in each S-phase.
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Stern, Bodo. "Control of G1 progression in fission yeast." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.264166.
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May, Karen Marie. "Molecular characterisation of fission yeast myosin II." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263251.
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Al-Harithy, Rowyda Nawwaf. "Characterization of the fission yeast rad2 gene." Thesis, University of Sussex, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239575.
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De, Sandip. "Tracking translation factors in fission yeast nucleus." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1421/.
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Translation factors are essential components of the ribosome, yet it has been reported that many ribosomal proteins (RPs) and other translation factors are found at transcription sites of Drosophila melanogaster (D. melanogaster) polytene chromosomes. Whilst these findings might indicate the presence of ribosomal subunits at transcription sites, it has also been reported that these proteins associate with non-coding RNA in Saccharomyces cerevisiae (S. cerevisiae), suggesting that their localization with transcription sites reflects their non-ribosomal function. However, the functional significance of RPs and translation factors at transcription sites is unclear and may reflect excess protein synthesize unincorporated into ribosomes, leading to a large pool of free proteins able to interact non specifically with other proteins and nucleic acids. The following work investigates these issues further in Schizosaccharomyces pombe (S. pombe). I tagged three RPs (RpL7, RpL11 and RpL25), by homologous recombination and used a Chromatin immunoprecipitation approach to investigate whether the association of RPs occurs across specific genes/transcripts or whether chromatin association is genome wide. In agreement with previous studies in D. melanogaster and S. cerevisiae, I found that RPs preferentially associate to transcriptionally active genes. ChIP followed by analysis on micro-arrays (ChIP-on-chip) revealed that RPs associate with several protein encoding genes. Further analysis of the three RPs showed that they tend to bind a common subset of genes. Whilst RNase sensitivity suggests RPs association with nascent RNA, I found no correlation between the ChIP-on-chip signals of RPs with either Pol II occupancy or transcript level but did show that RPs associate with non-coding-RNA genes most notably with tRNA genes. ChIP of RpL7 in a strain carrying an exogenous wild-type tDNATyr gene or a mutant with a non-functional promoter confirmed that RpL7 associates only to the active tRNA gene. These results suggest a functional role of RPs in tRNAs biogenesis and perhaps in a role in Pol III transcription, as it was recently suggested by the finding that RPs copurify with TFIIIE in S. cerevisiae. Nonsense-mediated mRNA decay (NMD) removes any mRNA containing premature termination codon (PTC), and requires Upf1. Phospho-Upf1 inhibits conversion of 40S/Met-tRNAiMet/mRNA to translationally competent 80S/Met-tRNAiMet/mRNA initiation complexes to repress continued translation initiation. Analysis with Upf1 showed Upf1 also associates with many transcription sites and has a role in DNA replication and/or repair. Notably, Upf1 binds to chromatin mostly during S phase; perhaps indicating a role for its helicase activity during DNA replication in S. pombe. In summary, my data indicate that association of RPs, and at least one NMD factor, to chromatin is a general feature of eukaryotes. The main challenge for future studies is to identify the factors driving this association and the functional significance.
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Lackner, Daniel H. "Genome-wide translational control in fission yeast." Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.611872.
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Dunleavy, Elaine. "Assembly of centromeric chromatin in fission yeast." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/13739.
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Fission yeast centromeres are composed of a central domain surrounded on both sides by outer repeat heterochromatin. Marker genes inserted within the central domain or the outer repeats are transcriptionally silenced. A screen performed to identify mutants that specifically alleviate silencing at the central domain, identified the sim (silencing in the middle of the centromere) mutants. The sim6 mutant was unusual in that it was found to alleviate both central domain and outer repeat silencing and suggests that there may be cross talk between kinetochore assembly and the integrity of neighbouring heterochromatin. To identify other factors with a similar phenotype a screen was performed in which the cos (central core and outer repeat silencing) mutants were isolated. Several mutants allelic to sim6+ (renamed cos1+) were isolated. Cos1+ is allelic to fission yeast mcl1+ (mini-chromosome loss 1) and may function to ensure that features of silent chromatin and sister chromatin cohesion are properly maintained after replication. sim3 mutants were found to specifically alleviate silencing at the central domain. Sim3 is homologous to the histone binding proteins mammalian NASP (nuclear autoantigenic sperm protein) and Xenopus laevis N1/N2. Cells with defective Sim3 have reduced levels CENP-ACnp1 at centromeres and subsequently display defects in mititotic chromosome segregation. Sim3 may be acting as a CENP-ACnp1 ‘escort’, assisting in the replication independent assembly of CENP-ACnp1 chromatin at centromeres, however Sim3 may also contribute to the loading of Sim3 at other stages of the cell cycle. It remains to be determined whether NASP/N1/N2 plays a similar role in metazoa.
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Sczaniecka, Matylda. "Analysis of anaphase inhibitors in fission yeast." Thesis, University of Edinburgh, 2007. http://hdl.handle.net/1842/14371.
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As a result of checkpoint activation, a signalling cascade is initiated and a number of complexes between the checkpoint components are formed. This leads to the inhibition of the Anaphase Promoting Complex (APC), which is the ubiquitin ligase responsible for targeting mitotic proteins: securing and cyclin B for degradation by the 26S proteasome. The complexes formed include the MCC, or Mitotic Checkpoint Complex, which in fission yeast (Schizosaccharomyces pombe) consists of Mad2, Mad3 checkpoint proteins together with the APC activator, Slp1 (the Cdc20 homologue). The MCC has been shown to bind and inhibit the APC in HeLa cells. In my PhD I focused on the interactions between the MCC and the APC, in particular on Mad3 protein. Mad3 is a conserved checkpoint component, homologous to human BubR1. It carries 2 putative KEN boxes, motifs, which typically target proteins for degradation (like D-boxes). We mutated both KEN boxes in S. pombe Mad3 and show that they are essential for Mad3 checkpoint function. One of the two motifs is also required for MCC formation, MCC/APC binding and Mad3 turnover in G1 stage of cell cycle. We argue that KEN boxes mediate the inhibitory interactions between checkpoint proteins and the APC. The formation of the MCC requires Mad2 and Mad3. These proteins are also dependent on one another for binding to the APC. We study the formation of the MCC, as well as its binding to the APC in different checkpoint mutants, trying to understand dependencies between these proteins and the requirement for kinetochore localisation. Finally, we make an attempt to study the cellular localisation of S1p1 and the APC and find that while S1p1 colocalises with a kinetochore marker, the APC subunits Cut9 and Lid1 remain distributed around the fission yeast nucleus.
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Almeida, Hugo Ricardo Noronha de. "Measuring chromosome-end fusions in fission yeast." Doctoral thesis, Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica, 2013. http://hdl.handle.net/10362/10629.
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Dissertation presented to obtain the Ph.D degree in Molecular BiologyThe ends of eukaryotic chromosomes are protected from illegitimate repair by structures called telomeres. These are comprised of specific DNA repeats bound by a specialized protein complex. When telomere function is compromised, chromosome ends fuse, generating chromosomal abnormalities and genomic instability.(...)
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Schmidt, Michael. "Regulation of protein turnover in fission yeast." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:93-opus-36654.
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Stone, Miranda Lucy. "Proteasome-associated deubiquitinating enzymes in fission yeast." Thesis, University of Edinburgh, 2002. http://hdl.handle.net/1842/23210.
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Two putative proteasome-associated DUBs were identified in S. pombe. Uch2 had previously been suggested to copurify and colocalise with the proteasome (Li et al., 2000). A second DUB, Ubp6, was identified in a sequence homology search for S. pombe proteins containing a ubiquitin-like (Ubl) domain (C. Semple and C. Gordon, unpublished). This domain has been shown to mediate interactions with the proteasome, implying that Ubp6 might also be proteasome-associated (Wilkinson et al., 2001). In the first part of this study, both Uch2 and Ubp6 are shown to be proteasome-associated DUBs. The S. pombe proteasome is purified and Uch2 and Ubp6 are both demonstrated to be present. In support of this finding, immunofluorescence microscopy reveals that Uch2 and Ubp6 colocalise with the proteasome at the nuclear periphery (Wilkinson et al., 1998). Construction of uch2 and ubp6 null mutants and the uch2ubp6 double mutant is described. These mutants do not have any obvious phenotype, suggesting that other redundant DUBs may be present at the proteasome. ubp6 is shown to be synthetically lethal with the mts1, mts2 and mts3 proteasome mutants, however uch2 is not synthetically lethal with any proteasome mutant tested (Gordon et al., 1993; Gordon et al., 1996; Penney et al., 1998; Wilkinson et al., 1997; Wilkinson et al., 2000, C. Gordon, unpublished). The DUBs function to cleave the αNH-peptide and the εNH-isopeptide bonds between ubiquitin and other species (Chung and Baek, 1999; Wilkinson, 2000). Purified 26S proteasomes and recombinant Uch2 are shown to cleave peptide linked ubiquitin using an in vitro assay and Uch2 is identified as the major ubiquitin hydrolase of the proteasome. Both Uch2 and Ubp6 are demonstrated to be capable of cleaving isopeptide-linked ubiquitin in vitro.
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Doe, Claudette Louise. "Characterisation of the fission yeast rad8 gene." Thesis, University of Sussex, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.357682.
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Edelmaier, Christopher. "Computational Modeling of Mitosis in Fission Yeast." Thesis, University of Colorado at Boulder, 2018. http://pqdtopen.proquest.com/#viewpdf?dispub=10837613.
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Mitosis ensures the proper segregation of chromosomes into daughter cells, which is accomplished by the mitotic spindle. During fission yeast mitosis, chromosomes establish bi-orientation as the bipolar spindle assembles, meaning that sister kinetochores become attached to microtubules whose growth was initiated by the two sister poles. This process includes mechanisms that correct erroneous attachments made by the kinetochores during the attachment process. This thesis presents a 3D physical model of spindle assembly in a Brownian dynamics-kinetic Monte Carlo simulation framework and a realistic description of the physics of microtubule, kinetochore, and chromosome dynamics, in order to interrogate the dynamics and mechanisms of chromosome bi-orientation and error correction. We have added chromosomes to our previous physical model of spindle assembly, which included microtubules, a spherical nuclear envelope, motor proteins, crosslinking proteins, and spindle pole bodies (centrosomes). In this work, we have explored the mechanical properties of kinetochores and their interactions with microtubules that achieve amphitelic spindle attachments at high frequency. A minimal physical model yields simulations that generate chromosome attachment errors, but resolves them, much as normal chromosomes do.
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Kristell, Carolina. "Chromatin Dynamics in the Fission Yeast, Schizosaccharomyces pombe." Doctoral thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-158084.
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In the eukaryotic cell nucleus, spatial organization and dynamics of the genome is important in the regulation of gene expression. This thesis describes the use of the fission yeast, Schizosaccharomyces pombe, to study chromatin regulation and dynamics. We used nitrogen starvation to induce transcription of genes in fission yeast cells. In induced genes, nucleosomes get evicted in both the promoter and in the open reading frame (ORF). In the genes with the highest expression more nucleosomes get evicted from the ORF than from the promoter. This indicates that large rearrangements of the chromatin are occurring during a drastic gene induction. Many of the genes that become expressed early after nitrogen starvation are located together in clusters. In a cell where nitrogen is present in the surrounding media the gene clusters locate close to the nuclear periphery. When the nitrogen source is removed from the media, the clusters move to a more internal position. Thus rearrangement of chromatin due to gene induction, described in the first study, is accompanied by subnuclear changes of localization. Another type of regulation is the silencing of genes. We have studied a factor necessary for correct repression of genes located in silent chromatin, in S. pombe. The protein, Clr2, is part of the SHREC complex containing a remodeler (Mit1) and a histone deacetylase (Clr3). By bioinformatic analysis of Clr2 and newly sequenced fungi genomes, three motifs were identified. To gather more information about important parts of the Clr2 protein, deletions were made. When removing from about 20 to 100 amino acids in the middle of the protein, silencing of a reporter gene inserted at the mating-type region, inner repeats of centromere 1 and at the central core of centromere 2, failed. This indicates that Clr2 has an important role in establishing silent chromatin.
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Fersht, N. "The checkpoint role of Cdc18 in fission yeast." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1445447/.
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The highly conserved eukaryotic checkpoints keep tight control over cell cycle progression, arresting the cell in response to incomplete DNA replication or DNA damage. In fission yeast, Rad3 (functional homologue of ATM, mutated in ataxia telangiectasia, and structural homologue of ATR, ataxia telangiectasia and rad3 related) is necessary for activation of both replication and damage checkpoints. However, despite the identification of many checkpoint genes, the actual sequence of upstream events leading to Rad3 activation remains unclear. The aim of my project was to identify and characterise the Rad3- dependent DNA damage/perturbed replication sensors and checkpoint activators. A genetic screen was carried out in fission yeast, using the working hypothesis that overexpression of these sensors/checkpoint activators would ectopically induce a Rad3-dependent block over mitosis in the absence of DNA damage or disturbed replication. The screen identified several genes of which the DNA replication initiation factor Cdc18/CDC6 had the strongest and most reproducible phenotype. Cdc18 is essential to prevent mitosis during S phase. I chose to concentrate on characterisation of the Rad3-dependent checkpoint role of Cdc18. The actual level of Cdc18 is important for producing the Rad3-dependent cell cycle block. A stabilised Cdc18 protein, mutated at the conserved CDK (cyclin dependent kinase) consensus sites also caused a Rad3-dependent cell cycle arrest, and was more stable and easier to manipulate than the screen- derived clone. Genetic crosses demonstrated Cdc18 acts early on in the checkpoint pathway, and through Crb2/Chk1. There was no gross DNA damage or detectable replication intermediates in the presence of elevated or stabilised Cdc18 levels. I also found that artificial depletion of Cdc18 during an S phase block results in loss of the checkpoint but not the replication structures, uncoupling the maintenance of replication forks from the maintenance of the mitotic block. An unexpected consequence of Cdc18 stabilisation was an increase in the size and variability of chromosome III on pulsed field gel electrophoresis. This localised to an expansion of Sfi1 restriction fragments containing the rDNA repeats. In conclusion, Cdc18 stabilisation activates a Rad3-dependent checkpoint in the absence of apparent re-replication, which is associated with an expansion of the rDNA repeats on chromosome III. Two models are proposed. In the first, Cdc18 induces low level genome wide replication, that is undetectable but sufficient for checkpoint activation. This leads to increased recombination with unequal crossover events in the rDNA repeats on chromosome III, with subsequent repeat expansion. In the second, the increased levels of Cdc18 directly activate the cell cycle checkpoint independently of the concurrent expansion of chromosome III.
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Raponi, Mitch Biochemistry & Molecular Genetics UNSW. "Antisense RNA-mediated gene silencing in fission yeast." Awarded by:University of New South Wales. Biochemistry and Molecular Genetics, 2001. http://handle.unsw.edu.au/1959.4/18277.
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The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.
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Bond, Michael Edward. "Ras signalling in the fission yeast Schizosaccharomyces pombe." Thesis, University of Warwick, 2012. http://wrap.warwick.ac.uk/46013/.
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Ras signalling is vital to many cellular processes. Ras proteins mediate a vast array of cellular signalling networks, and are conserved from humans to unicellular eukaryotes. The study of ras signalling in higher eukaryotes presents a number of technical challenges, due to the presence of multiple ras isoforms, regulatory proteins and activators. The fission yeast Sz. pombe represents an ideal system for the investigation of ras signalling, as it contains a single, nonessential ras protein (Ras1). In addition, Ras1 is involved in the regulation of a number of downstream pathways. A number of studies in recent years have highlighted the role of subcellular localisation in ras signalling output. The localisation of Ras1 in Sz. pombe has also been described as key in effector selection, with Ras1 at the plasma membrane regulating mating and Ras1 at the endomembranes regulating cell morphology. This thesis describes a series of studies utilising Ras1 mutants and chimeric Ras1 proteins which display differing localisation patterns to determine the role of Ras1 localisation in signalling. The data presented herein support the notion of a revised model for the role of Ras1 localisation in signalling, suggesting that the localisation of Ras1 to the plasma membrane is key to all signalling events downstream of Ras1. This thesis also describes the characterisation of oncogenic mutants of Ras1, demonstrating the importance of signalling magnitude in functional output. In addition, the importance of Ras1 regulation in cell viability and chromosome stability is also demonstrated. Finally, the functional expression of three human ras isoforms is described, validating the use of Sz. pombe as a model system for the heterologous expression of human ras signalling components.
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Schmidt, C. K. "Genomic analysis of cohesin dynamics in fission yeast." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/14917/.
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Cohesin holds sister chromatids together and facilitates their accurate segregation in mitosis. Little is known about how and where cohesin binds to chromosomes. Recent genome-wide investigations have led to apparent disparities between different model organisms. In this thesis, analysis of the cohesin binding pattern reveals that several determinants, thought specific for distinct organisms, collectively define the overall distribution of cohesin along fission yeast chromosomes. Like in budding yeast, cohesin is mainly detected at sites of convergent transcriptional termination, in the following termed convergent sites. However, only approximately half of these are bound whereas in budding yeast almost all of them are associated with cohesin. Furthermore, we detect cohesin at loci away from convergent sites which are characterised by the presence of the cohesin loader Mis4/Ssl3. Cohesin loading sites show a striking overlap with strongly transcribed genes, including tRNA and ribosomal protein genes. This is reminiscent of Drosophila cohesin and its loading factor Nipped-B that both overlap near highly transcribed genes. The cohesin loader also promotes cohesin accumulation at neighbouring convergent sites, which, together with gene arrangement and transcription, contributes to the distribution of cohesin among convergent sites. Cohesin binding to G1 chromosomes depends on the continuous activity of the cohesin loader Mis4/Ssl3. Cohesin stability then increases during S phase independently of DNA replication but in part dependent on the acetyltransferase Eso1, a factor implicated in the establishment of cohesion. This indicates that cohesin stabilisation might be a pre-requisite for cohesion establishment rather than its consequence. During mitosis, a fraction of cohesin leaves chromosomes in a cleavage-independent reaction in prophase similarly to what has been observed in higher eukaryotes. A substantial pool of cohesin then dissociates from chromosomes upon its cleavage at anaphase onset. As a unique feature, centromeric cohesin spreads out onto chromosome arms towards anaphase as the heterochromatin protein Swi6 dissociates from centromeres. Taken together, our results suggest conserved mechanisms for both cohesin binding and dynamics across eukaryotes.
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Hogan, C. "Functional characterisation of the fission yeast Ino80 complex." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.604143.
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INOsitol-requiring 80 (Ino80) is a catalytic ATP-dependent nucleosome remodelling enzyme of the Ino80 complex which is involved in transcription, replication and the DNA damage response. In this thesis, I characterise for the first time the Ino80 complex from Schizosaccharomyces pombe. Purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein, lec1, with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this lec1 protein or the Ino80 complex subunits: Arp8, les6 or les2 causes defects in DNA damage repair, response to replication stress and nucleotide metabolism. I demonstrate that lec1 is important for the correct expression of genes involved in nucleotide metabolism such as ribonucleotide reductase subunit cdc22. Ino80 is recruited to a large number of promoter regions upon phosphate starvation, including those of phosphate and adenine responsive genes that depend on lec1 for correct expression. lec1 is required for binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes. this suggests that the lec1-Ino80 complex promotes transcription through nucleosome eviction. My study of the Ino80 complex subunits lec1 and Arp8 shows that they act together or oppose one another, depending on the target locus. These subunits affect gene expression, histone density and deposition of histone marks: H3K4me3, H3K9me3 and H3K36me3. Arp8 affects iec1 gene expression and regulates the presence of lec1 at target genes. These results reveal the modular nature of the Ino80 complex. Finally, I show that deletion of Ino80 subunits leads to sensitivity to drugs that inhibit transcription elongation.
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Amoah-Buahin, Evelyn. "Hyphal growth in the fission yeast Schizosaccharomyces pombe." Thesis, University of Sussex, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430364.
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Baum, Benjamin. "Control of S-phase transcription in fission yeast." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265313.
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Samuel, Margaret Jane. "Novel regulators of the fission yeast cell cycle." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272174.
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Riaz, Abida. "Cyclin B in fission yeast mitosis and meiosis." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286160.
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Behrens, Ralf. "Molecular mechanisms of cell polarity in fission yeast." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.271825.
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Armes, Helen Elizabeth Harcourt. "Implementing super-resolution palm microscopy in fission yeast." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/67027/.
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Fluorescence microscopy is a popular biological technique because it allows the study of cells in great detail. However, the resolution achievable is limited by the diffraction properties of light, meaning that fine detail cannot be resolved. Various super-resolution microscopy methods have been developed to break this resolution limit. This thesis focuses on the single molecule localisation microscopy techniques. My host laboratory focuses on DNA replication and repair pathways using the model organism Schizosaccharomyces pombe (fission yeast). The aim of this thesis is thus to apply the technique of photo-activatable localisation microscopy (PALM) to specific biological questions in order to establish its benefits and limitations. In theory, in PALM every molecule will be imaged once and, as such, could be counted. So far this has been largely limited to membrane proteins. Using a combination of artificially created fluorescent oligomers, endogenous ribonucleotide reductase proteins tagged with mEos and computer simulations I studied the feasibility of counting highly expressed cytoplasmic proteins and assigning them to complexes of known or unknown stoichiometry. I established that density of expression is a significant limiting factor when using PALM to resolve complex stoichiometry. I thus went on to develop a variation of fluorescence correlation spectrometry to study the same protein complexes to see if we could determine their stoichiometry by diffusion speed. I established that the technique could differentiate between quite small changes in size. However the endogenous complex did not respond well to the fluorophore used so I was not able to establish its size. Using the PALM system I also studied a biological molecule, Rrp2, which was expressed at such low levels it was not possible to observe with conventional fluorescence microscopy. I established that we were able to observe this protein at endogenous levels and characterised its behaviour in response to stress.
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Abraham, Anne. "Polo-like kinase interacting proteins in fission yeast." Thesis, University of Edinburgh, 2004. http://hdl.handle.net/1842/10699.
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Kms1 and Kms2 are important for integrity of the SPB. To find the biological significance of the interactions between Plo1 and these SPB proteins, attempts were made to disrupt the interaction by mutations. For this purpose, firstly regions responsible for the interaction were identified, and then mutations were made in the SPB proteins by random mutagenesis of this region. For Sid4, I isolated two point mutations, which had greatly weakened interaction with Plo1. To study the effect of disrupting the interaction in vivo, the two sid4 mutants were expressed from a fission yeast promoter in the sid4 temperature-sensitive mutant. Both point mutants of Sid4 that had weakened interaction with Plo1 were able to rescue the temperature-sensitive sid mutant with a similar strength as that of wild-type sid4 under the same promoter. The identification of potential Plo1 interacting proteins and mutants defective in these interactions will be an important step to understand cell cycle control by Plo1.
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Almeida, Ricardo. "RNA interference and heterochromatin formation in fission yeast." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/11198.
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There is strong evidence suggesting that RNAi acts co-transcriptionally in order to promote heterochromatin formation. Thus, it is possible that RITS activity is interlinked with transcription-related processes such as cleavage/poly-adenylation, transcription termination and RNA turnover by the exosome complex. In order to investigate this hypothesis, the integrity of RNAi and heterochromatin was assayed in mutants for factors that are involved in all three pathways. Mutations on dhp1 (termination), pfs2 (cleavage and polyadenylation) dis3 and rrp6 (exosome) had negligible effects on RNAi activity and heterochromatin-mediated silencing with only the exosome showing some involvement in the downstream degradation of centromeric transcripts. Conventional RNAi enforces post-transcriptional repression by targeting mRNA molecules for degradation. This is mediated by the endonuclease activity of Argonaute (“slicing”). Although the key residues for this activity are conserved between human Ago2 and S. pombe Ago1, the importance of this “slicing” activity to heterochromatin assembly was not clear. Mutations were made in putative catalytic residues on the endogenous ago1 gene in order to address this question. These mutations severely affect the activity of RNAi in fission yeast and destabilize the heterochromatin structure at centromeres. Consequently, centromere function is affected and chromosome segregation is deficient. Ago1 localization to the centromeres is impaired in these mutants and cannot nucleate heterochromatin nucleation though siRNA production is not fully abolished. Thus, Ago1 slicing activity is crucial for sustainable RNAi and its role in heterochromatin integrity.
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McInerny, Christopher. "Control of S-phase genes in fission yeast." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/12615.
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The work described in this thesis uses the fission yeast Schizosaccharomyces pombe as model system to analyse the molecular processes that control passage through the G1 and S-phases of the cell-cycle. In particular, the work analyses the control of expression of cdc22+, a gene whose transcript varies in abundance during the cell cycle with a maximum at the G1-S phase boundary. Chapter two describes the sequencing of cdc22+ and shows it encodes the large subunit of ribonucleotide reductase, an enzyme required for DNA precursor metabolism. In the next Chapter, cis-acting elements, resembling MCBs previously identified in budding yeast, are shown to be present 5' to the cdc22+ open reading frame. MCBs can confer cell cycle expression on a heterologous gene in fission yeast, implicating them in controlling periodic expression of cdc22+. A trans-acting complex that specifically binds MCBs is identified, and called DSP1-for b DNA b synthesis control in S.b pombe. DSP1is related to DSC1, an MCB binding activity identified in budding yeast. Experiments in Chapter 3 demonstrate that the gene product of the cdc10+ START gene, p87cdc10, isa component of DSP1. Furthermore, cdc22+ is shown to be constitutively over-expressed in a cdc10 mutant, cdc10-C4. Over-expression of cdc22+ in cdc10-C4 is recessive to wild-type. It is proposed that DSP1 containing p87cdc10-C4 is hyperactive and deregulated as a transcription complex. In summary, p87cdc10 is part of a transcription complex that controls expression of cdc22+, agene required for DNA synthesis. Thus this work demonstrates a molecular link between START and S-phase.
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Lord, Phillip. "Genes affecting the centromeres of the fission yeast." Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/15235.
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In the fission yeast, S. pombe the centromeres have been extensively characterised, and consist of a largely unique central core, surrounded by a large inverted repeat. Previous studies have shown that marker genes inserted into these regions are transcriptionally silenced. Several genes have been isolated which are required for this transcriptional silencing. One of these, swi6*, has been shown to localise to the centromere in vivo and it thought to form part of the functional kinetochore. In this thesis I describe the first molecular characterisation of another gene, clr4* which is also required for centromeric silencing, and for localisation of Swi6p to the centromere. This gene and several of its alleles have been fully sequenced, and shown to contain both a chromo-domain and a SET domain. This pattern of domain organisation has previously been shown to occur in the Drosophila gene Suvar (3)9, indicating that the silencing processes in this organisms are similar to those seen in S. pombe. Four of the mutant alleles sequenced result from point mutations, and of these, three alter conserved residues in the SET domain. The clr4* gene was disrupted leaving only 38 amino acids at the 5' end of the native open reading frame intact. This disruption was shown to have a phenotype similar or identical to that of the originally isolated clr40s5 allele. I also describe the characterisation of a novel gene, eval+ which was isolated due to its effect on transcriptional silencing of reporter genes placed within the central core of the S. pombe centromeres. This gene has been fully sequenced and shown to contain a SANT domain. Possible functions for this gene in the cell are discussed.
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Richardson, Kathryn. "Mechanisms of GPCR signal regulation in fission yeast." Thesis, University of Warwick, 2014. http://wrap.warwick.ac.uk/63554/.
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Cells communicate with each other and respond to environmental cues by sending and receiving signals. Many external signals (ligands) are detected through G protein-coupled receptors (GPCRs), a major class of transmembrane proteins. GPCRs transduce these external signals into appropriate intracellular responses, enabling the cell to adapt to its environment. Malfunctions in these signalling pathways can lead to a range of human diseases and hence GPCRs have become attractive candidates for pharmacological design. The activation of a single receptor has the ability to induce numerous intracellular responses. Coupling this with the great number of different GPCR-types expressed in human cells means that understanding the basic principles of signal transduction and termination in humans is complicated. This study utilises the more simplistic eukaryotic yeast Schizosaccharomyces pombe (S. pombe) to overcome this complexity, as it contains only two GPCR types and hence the cross-talk between pathways is greatly reduced, whilst the structure and signalling functions of GPCRs are often evolutionarily conserved between yeast and humans. Mathematical modelling was used to aid the understanding of GPCR signalling in S. pombe and to inform experimental design. Speci�cally, an ordinary differential equation model �rst developed by Croft et al. (2013) was extended to include all known downstream signal transduction, regulation and termination events. This model is the �rst of its kind to describe a whole GPCR signalling pathway within S. pombe. Although it accurately predicts the cellular response to GPCR signalling it could only reproduce the biological plateau in temporal response with the addition of a 'yet unknown mechanism' GPCR degradation term. This motivated the investigation of how GPCRs in S. pombe are internalised from the plasma membrane in response to ligand stimulation. The primary mechanism for signal termination is via internalisation of the GPCR. This study identi�ed three potential casein kinases (Cki1, Cki2 and Cki3) that promote internalisation of the S. pombe GPCR Mam2. Microscopy analyses in combination with quantitative transcriptional, cell growth and cell cycle position assays uncovered a novel role for these kinases: that Cki2 regulates cell size during vegetative growth, Cki1 and Cki3 regulate the GPCR-response pathway and that Cki3 is essential for completing cytokinesis in S. pombe that have already undergone formation of a conjugation tube in response to ligand. Confocal microscopy of uorescent labelled Mam2 indicated a role for Cki2 in the internalisation and hence termination of the GPCR-response pathway. These findings add to the growing body of evidence that casein kinases are implicated in GPCR desensitisation.
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Cantwell, Helena Rose. "Nuclear size control and homeostasis in fission yeast." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10054466/.
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Despite it being recognised as a problem worthy of consideration over a century ago, we have little mechanistic understanding of how the size of a cell’s nucleus is determined. The simply shaped fission yeast Schizosaccharomyces pombe is genetically tractable and undergoes a closed mitosis, making it a useful system in which to probe mechanisms of nuclear size control. In S. pombe cells, nuclear volume scales with cell volume, and not DNA content, across a wide range of cell volumes and throughout the cell cycle, maintaining a constant nuclear volume to cell volume (N/C) ratio. This thesis explores the mechanisms by which this scaling is achieved, using physiological, genetic and biochemical approaches. N/C ratio is perturbed and resultant nuclear and cellular growth rates of individual cells are assessed. N/C ratio homeostasis is observed. Both high and low aberrant N/C ratios correct rapidly in individual cells. Analysis of the kinetics of N/C ratio recovery is carried out and mathematical models of nuclear size control are proposed. To identify molecular components and biological processes with roles in nuclear size control mechanisms, a genetic screen for deletion mutants with aberrant nuclear size and biochemical analysis of a nuclear size mutant are carried out. Ribosome biogenesis, RNA processing and nucleocytoplasmic transport are all implicated in nuclear size control.
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Feret, Dorota. "Proteasomal control of the fission yeast microtubule cytoskeleton." Thesis, University of Manchester, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.704739.
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Patch, Ann-Marie. "A comparative analysis of tandem repeats in the fission yeast and budding yeast genomes." Thesis, University of Exeter, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425493.
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Dolan, William P. "Molecular genetic analysis of the fission yeast hsk1⁺ kinase /." Diss., Connect to a 24 p. preview or request complete full text in PDF formate. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3189214.
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Yin, Ling. "Activation of DNA Replication Initiation Checkpoint in Fission Yeast." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/194.
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In the fission yeast, Schizosacchromyces pombe, blocks to DNA replication elongation trigger the intra-S phase checkpoint that leads to the activation of the Cds1 kinase. Cds1 is required to both stabilize stalled replication forks and to prevent premature entry into mitosis. Interestingly, although Cds1 is essential to maintain the viability of mutants defective in DNA replication elongation, my study shows that mutants defective in DNA replication initiation require the Chk1 kinase, rather than Cds1. This suggests that failed initiation events can lead to activation of the DNA damage checkpoint independent of the intra-S phase checkpoint. This might result from reduced origin firing that leads to an increase in replication fork stalling or replication fork collapse that activates the G2 DNA damage checkpoint. I refer to the Chk1-dependent, Cds1-independent phenotype as the rid phenotype (for replication initiation defective). The data shows that Chk1 is active in rid mutants when grown under semi-permissive conditions, and rid mutant viability is dependent on the DNA damage checkpoint, and surprisingly Mrc1, an adaptor protein required for activation of Cds1. Mutations in Mrc1 that prevent activation of Cds1 have no effect on its ability to support rid mutant viability, suggesting that Mrc1 has a checkpoint-independent role in maintaining the viability of mutants defective in DNA replication initiation. Like Mrc1, Swi1 and Swi3 have been hypothesized as a part of the replication fork protection complex (RFPC). They are required for maintaining the viability of rid mutants, but are not essential for activation of Chk1 in response to failed initiation events. This suggests that Mrc1 in conjunction with Swi1 and Swi3 function in a similar pathway to alleviate replicative stress resulting from defects in DNA replication initiation. Using flow cytometry, I demonstrate that inhibition of DNA replication initiation has no significant impact on the duration of S phase, suggesting dormant origins might be activated in response to defects in DNA replication initiation. Fission yeast Rad22 is implicated in forming nuclear foci in response to damaged DNA. By tracking YFP-labeled Rad22, I screened for potential DNA damage in rid mutants grown at semi-permissive temperatures, and the results show that DNA damage occurs as the result of defects in DNA replication initiation. I also identified camptothecin, a DNA topoisomerase I inhibitor that can at low dose (2 µM) induce the rid phenotype, suggesting our assay (Chk1-dependent, Cds1-independent) can be used to screen small molecule inhibitors that interfere with the initiation step of DNA replication.
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Labib, Karim. "Regulation of S-phase and mitosis in fission yeast." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.358653.
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Ors, Aslihan. "Identification of novel APC/C regulators in fission yeast." Thesis, Institute of Cancer Research (University Of London), 2009. http://publications.icr.ac.uk/10297/.
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Sequential degradation of cell cycle proteins is a major regulatory mechanism for proper cell proliferation. Turnover of these proteins is performed by the ubiquitin/proteasome pathway, which poly-ubiquitylates and subsequently degrades the target proteins. The anaphase-promoting complex/cyclosome (APC/C) is a multi subunit E3 ubiquitin ligase that provides substrate specificity and regulation of the ubiquitin pathway. Two of the most important substrates are Cut2/securin and Cdc13/cyclin B, enabling chromosome segregation and exit from mitosis, respectively. Since APC/C function is very important for the cell cycle progression, its activity is strictly regulated. The aim of this study is to understand the regulation of APC/C and how its subunits contribute to this regulation. For this purpose, we have focused on the two least understood subunits: Apc1/Cut4 and Apc5. Conditionally lethal temperature sensitive mutants of these two subunits were created in fission yeast and the used for suppressor screening. We identified atf1+ as a multicopy suppressor of apc5-1, a mutation causing mitotic arrest. Fission yeast Atf1, which is homologous to human ATF2/CRE-BP1, is a bZIP domain mutant of Atf1, which has lost its transcription activation function, was still able to suppress the ts phenotype of apc5-1. The atf1+-dependent rescue was specific to the apc5-1 allele, rather than rescuing other APC/C subunit mutants and deletion of atf1+ increased the mitotic defects of the mutant. Interestingly, Atf1 physically binds to the APC/C in vivo. Furthermore, in vitro studies proved that Atf1 stimulates ubiquitylation activity of the APC/C. Finally; this interaction was shown to contribute to conjugation/mating efficiency of S. pombe cells upon nitrogen starvation and G1 arrest. Altogether, these results reveal a novel role for Atf1 in regulating the APC/C ubiquitin ligase, besides its transcription factor activity.
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Ananthanarayanan, Vaishnavi. "Dynein dynamics during meiotic nuclear oscillations of fission yeast." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-135620.
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Cytoplasmic dynein is a ubiquitous minus-end directed motor protein that is essential for a variety of cellular processes ranging from cargo transport to spindle and chromosome positioning. Specifically, in fission yeast during meiotic prophase, the fused nucleus follows the spindle pole body in oscillatory movements from one cell pole to the other. The three molecular players that are essential to this process are: (i) the motor protein dynein, which powers the movement of the nucleus, (ii) microtubules, which provide the tracts for the movement and (iii) Num1, the anchor protein of dynein at the cortex. Dyneins that are localized to the anchor protein at the cortex and simultaneously bound to the microtubule emanating from the spindle pole body, pull on that microtubule leading to the movement of the nucleus. The spindle pole body, by virtue of its movement establishes a leading and a trailing side.
Previous work by Vogel et al. has elucidated the mechanism of these oscillations as that of asymmetric distribution of dynein between the leading and trailing sides. This differential distribution is a result of the load-dependent detachment of dynein preferentially from the trailing microtubules. This self-organization model for dynein, however, requires a continuous redistribution of dynein from the trailing to the leading side. In addition, dyneins need to be bound to the anchor protein to be able to produce force on the microtubules. Anchored dyneins are responsible for many other important processes in the cell such as spindle alignment and orientation, spindle separation and rotation. So we set out to elucidate the mechanism of redistribution of dynein as well as the targeting mechanism of dynein from the cytoplasm to cortical anchoring sites where they can produce pulling force on microtubules.
By employing single-molecule observation using highly inclined laminated optical sheet (HILO) microscopy and tracking of fluorescently-tagged dyneins using a custom software, we were able to show that dyneins redistributed in the cytoplasm of fission yeast by simple diffusion. We also observed that dynein bound first to the microtubule and not directly to the anchor protein Num1. In addition, we were able to capture unbinding events of single dyneins from the microtubule to the cytoplasm. Surprisingly, dynein bound to the microtubule exhibited diffusive behaviour. The switch from diffusive to directed movement required to power nuclear oscillations occurred when dynein bound to its cortical anchor Num1. In summary, dynein employs a two-step targeting mechanism from the cytoplasm to the cortical anchoring sites, with the attachment to the microtubule acting as the intermediate step.
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Smith, Matthew William. "Studies of cyclins in the fission yeast Schizosaccharomyces pombe." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266483.
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