Halle, Universiẗat, Diss., 2001.

Universiẗat, Diss., 2004--Düsseldorf.

Meiosis is the process by which sexually-reproducing organisms ensure that precisely half a chromosome set is passed from each parent to the following generation; this circumvents the doubling of the genome that would otherwise occur upon fertilisation. Meiosis occurs via a single round of DNA replication followed by two successive chromosome segregation events. In the first segregation, homologous chromosomes align and become physically linked through the process of meiotic recombination, which is crucial for the accurate segregation of homologous chromosomes. During the second round of segregation, sister chromatids are segregated to produce four haploid daughter cells. Failure to physically tether homologous chromosomes to each other through meiotic recombination can result in the aberrant segregation of homologous chromosomes, which can cause hereditary diseases (aneuploidies) and miscarriages in humans. Meiotic recombination also shuffles alleles of the parental chromosomes, which is crucial for evolution. The study of meiotic recombination, and its regulation, is thus paramount for our understanding of how genetic diversity is generated within populations. The work in this thesis has helped characterise factors, both genetic and environmental, that modulate meiotic recombination in the fission yeast, Schizosaccharomyces pombe. Here, I identify temperature as a major determinant of meiotic recombination outcome; when meiosis is performed at 16°C, significant reductions in meiotic recombination outcome are observed relative to meiosis performed at higher temperatures. Additionally, I present genetic and cytological evidence that the strand resection and strand invasion steps of meiotic recombination are impaired at 16°C relative to higher temperatures, but that double strand break levels appear not to be influenced by temperature. I have also characterised several novel genes predicted to be involved in meiotic recombination, and explored the genetic relationship between several genes already known to be crucial in modulating meiotic recombination. Finally, I have laid the foundations for a future project aiming to map the meiotic recombination landscape across the entire S. pombe genome.

No description available.

No description available.

Les protéines d'interférence par l'ARN (RNAi) chez S. pombe sont responsables de la formation dliétérochromatine aux centromeres durant la mitose via le complexe RTTS -spChpl spTas3 spAgol-. De façon intéressante, la mutation d'une des protéines de RNAi, soit spAgol, spDcrl ou spRdpl, conduit à des aberrations de ségrégation chromosomique en méiose. Bien que ce phénotype puisse être dû à un défaut des centromeres, nous nous sommes intéressés à identifier une possible implication des protéines du RNAi dans un autre mécanisme primordial en méiose : la recombinaison homologue. Nous avons étudié les interactions potentielles entre les protéines de RNAi et les protéines de recombinaison homologue. Nous avons inclus également spArbl, spArb2, spChpl et spTas3, quatre protéines impliquées dans la formation dliétérochromatine aux centromeres via le RNAi. Nous avons ainsi démontré par double-hybride la présence d'interaction entre la protéine méiotique spRecl5 et spRdpl, spArbl et spChpl d'une part, et spRec7 avec spTas3 d'autre part. La deletion de ces gènes conduit à de graves défauts dans la formation des spores, soulignant leur importance dans le processus méiotique. spRec7 et spRecl5 sont deux protéines peu connues, impliquées dans les étapes précoces de la formation des cassures double brin par Spol 1 (spRecl2) à l'origine de la recombinaison homologue. Nous avons analysé par immunoprecipitations de chromatine et par immunofluorescence l'effet des mutations de spChpl et spTas3 sur les étapes précoces de recombinaison homologue, et les résultats préliminaires obtenus à ce jour semblent supporter une forte implication de ces protéines dans la recombinaison homologue méiotique.

Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: The fission yeast Schizosaccharomyces pombe is able to effectively degrade extracellular L-malate by means of a permease for the active transport of L-malate and a malic enzyme that catalyses the intracellular oxidative decarboxylation of L-malate to pyruvate and CO2. Sequence analysis of the S. pombe NAD-dependent malic enzyme gene, mae2, revealed an open reading frame of 1695 nucleotides, encoding a polypeptide of 565 amino acids. Mutational analyses of the mae2 promoter region revealed several putative cis-acting elements. Two of these elements have homology with binding sites for eukaryotic cAMPdependent regulatory proteins. The UAS I showed homology with the invert of the ADRI binding site, an AP-2 binding site and the TGGCA element. The other putative cAMPdependent site, UAS2, showed homology with the binding site for ATF/CREB and proved to be a strong activator sequence that is required for expression of the mae2 gene. Three negative acting elements, DRS I, DRS2 and DRS3 seem to function co-operatively to repress transcription of the mae2 gene. In this study northern and western blot analyses, as well as malic enzyme assays, showed increased levels of mae2 transcription and enzyme activity when cells were grown under fermentative conditions. The levels of mae2 expression increased approximately 4-fold in 30% glucose and 3-fold under anaerobic conditions. These increased levels of malic enzyme may provide additional pyruvate for various metabolic processes when the mitochondria are not fully functional under fermentative conditions. The regulated expression of the mae2 gene was further investigated using mae2-1acZ fusion plasmids that carried mutations in the DASI, UAS2 or the triple mutated DRSI/URS2/URS3 elements. These plasmids were transformed into S. pombe strains with mutations in the cAMP-dependent or stress-activated signal transduction pathways to determine the signal for the increased expression of the mae2 gene. The cAMP-dependent (Pkal ) and general stress activated (Styl) pathways often act in parallel to regulate the activation of transcription factors necessary for the expression of several S. pombe genes under different physiological conditions. The results presented here suggest that regulatory proteins involved in the Pka l and Styl pathways play a role in the regulation of the mae2 gene under fermentative conditions. Furthermore, some of the regulatory cis-acting elements in the mae2 promoter may interact with these trans-acting factors to regulate the transcription of the gene under different growth conditions. The mechanism of this interaction is not yet known and further research is required to identify all the transcription factors involved in the regulation of the mae2 gene. / AFRIKAANSE OPSOMMING: Die splitsingsgis S. pombe is in staat om ekstrasellulêre L-malaat effektief af te breek danksy 'n permease vir die aktiewe opname van L-malaat en 'n malaatensiem wat die intrasellulêre oksidatiewe dekarboksilering van L-malaat na pirovaat en C02 kataliseer. DNA-geen opeenvolgings van die NAD-afhanklike malaatensiemgeen, mae2, het 'n oopleesraam van 1695 nukleotiede getoon wat vir 'n polipeptied van 565 aminosure kodeer. Mutasie-analise van die mae2-promoter gebied het verskeie moontlike cis-werkende elemente getoon. Twee van die elemente toon homologie met bindingsetels vir eukariotiese cAMP-afhanklike regulatoriese proteïene. Die DAS 1 toon homologie met die omgekeerde volgorde van die ADRI bindingsetel, 'n AP-2 bindingsetel en 'n TGGCA element. Die ander moonlike cAMP afhanklike setel, DAS2, toon homologie met die bindingsetel vir ATF/CREB en is 'n sterk aktiveringselement wat vir die uitdrukking van die mae2-geen benodig word. Drie onderdrukker-tipe elemente, DRSI, DRS2 en DRS3, funksioneer moontlik gesamentlik om die transkripsie van die mae2-geen te onderdruk. In hierdie studie het northern en western klad analise, sowel as malaatensiem aktiwiteitstoetse verhoogde vlakke van mae2-transkripsie en ensiemaktiwiteit getoon wanneer die kulture onder fermentatiewe toestande gegroei het. Die uitdrukking van die mae2-geen het ongeveer 4-voudig toegeneem in 30% glukose en 3-voudig onder anaërobiese toestande. Hierdie verhoogde uitdrukking van die malaatensiem mag addisionele pirovaat vir verskeie metaboliese behoeftes voorsien wanneer die mitochondria onder fermentatiewe toestande nie volkome funksioneer nie. Die uitdrukking van die mae2-geen is verder onder fermentatiewe toestande bestudeer deur gebruik te maak van mae2-lacZ-fusie plasmiede wat mutasies in die moontlike DASI, DAS2, of die drievoudig-gemuteerde DRS I/URS2/URS3 setels bevat. Hierdie plasmiede is in S. pombe rasse met mutasies in die cAMP-afhanklike of stres-geaktiveerde seintransduksie paaie getransformeer om die sein vir die verhoogde mae2-geen uitdrukking te bepaal. Die cAMP-afhanklike (Pkal) en algemene stres-aktiverings (Styl) pad werk soms in parallel om die aktivering van transkripsiefaktore betrokke in die uitdrukking van verskeie S. pombe gene onder verskillende fisiologiese toestande to bewerkstellig. Ons resultate dui daarop dat die regulatoriese proteïene van die Pkal en die Styl paaie 'n rol in die regulering van die mae2- geen onder fermentatiewe toestande speel. Daar is ook aanduidings dat sommige van die regulatoriese cis-werkende elemente in die mae2-promoter wisselwerking met die transwerkende faktore toon om die transkripsie van die geen onder verskillende groeitoestande te reguleer. Die meganisme van hierdie interaksie is nog nie bekend nie en verdere navorsing is nodig om al die transkripsiefaktore wat by die regulering van die mae2-geen betrokke is, te identifiseer.

Thesis advisor: Charles Hoffman / The study of mammalian cAMP signaling has often been confounded by the fact that ten different genes encode adenylyl cyclases (ACs) that produce cAMP from ATP and 16 different genes encode phosphodiesterases (PDEs) that hydrolyze cAMP to AMP. In this study, mammalian AC cDNAs were cloned and integrated into strains of the fission yeast Schizosaccharomyces pombe that lack their endogenous AC to determine the basal activity of all ten AC isoforms. In addition, response to the stimulatory mammalian Gsα was determined by co-expression of a mutationally-activated form of the human GNAS1 gene. AC activity was assessed using an fbp1-GFP reporter that is repressed by cAMP production and PKA activity. Results confirm that all ten isoforms have detectable basal activity, and AC1-9 definitively respond to Gsα stimulation. When matched with a sufficiently potent mammalian phosphodiesterase (PDE), strains expressing mammalian ACs make good candidates for small molecule high throughput screening (HTS) to detect AC inhibitors. A 100,000 compound screen was recently performed to detect AC and Gsα inhibitors as well as PDE activators. A promising “hit” was progesterone, which has been previously suggested to inhibit ACs in Xenopus. Initial results suggest that progesterone inhibits AC1 and the closely-related AC3. These data demonstrate the utility of using S. pombe as an effective platform for identifying inhibitors of both basal and GNAS1-stimulated AC activity. / Thesis (BS) — Boston College, 2015. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Cell polarity is essential for cellular functions, growth, development, and formation of multicellular organisms. Cell polarization is often regulated during the cell division cycle. For instance, many cell types lose polarity and round up during mitosis, and then reestablish polarity after division. The fission yeast Schizosaccharomyces pombe is a model system for studying cell polarization. These unicellular rod-shaped cells grow by extension from their tips, and then stop growth during mitosis. Upon cytokinesis, they initiate growth from the old cell end and later in interphase, initiate growth at the second cell end in a process known as "new end take off" or NETO. NETO is regulated by polarity proteins tea1p and tea4p which are deposited by microtubules at the cell tips. How these proteins regulate cell polarity is not yet well understood. These polarity proteins are thought to function in recruiting other proteins, which leads to localized actin polymerization, membrane trafficking and cell wall assembly, leading ultimately to polarized cell growth at the cell tip. In this thesis, I report the characterization of a new polarity protein tea5p in fission yeast. I identified tea5p in a screen for new NETO mutants. Tea5p is a new component of the tea-protein polarity pathway. It resides at cell tips in complexes with the other polarity proteins tea1p and tea3p, and functions downstream of tea1p. Genetic interactions suggest that tea5p regulates polarized growth by regulating the small GTPase cdc42p and its activator gef1p. Tea5p is a pseudokinase that binds to the plasma membrane with its N terminus, and requires its kinase like domain for function. Together my results begin to establish a pathway that links microtubules to activation of cdc42p for regulation for polarized growth in S. pombe.

In order to contain many millions, or even billions of base pairs within every nucleus of a eukaryotic cell, DNA must be extensively packaged. This is achieved by association of DNA with packaging proteins, resulting in the formation of chromatin, which can lead to various degrees of compaction. The most extreme form of compaction is the highly condensed mitotic chromosome, formation of which is necessary for proper resolution and segregation of the genetic material during cell division. However, the exact nature of the structure of chromatin within the mitotic chromosome and the factors which regulate it remain subjects of debate and continued investigation. The hybrid cell line F1.1 presents a unique tool for the study of mitotic chromosome structure. This mouse cell line has been observed to present a distinct chromatin structure in mitosis assembled over a large region of DNA inserted into one of its chromosomes and originating from the fission yeast Schizosaccharomyces pombe. Direct comparison of the structure of this distinct region of chromatin with that of the adjacent endogenous chromatin could provide insight into the nature of mitotic chromosome structure as well as the properties of the chromatin which are influencing this structure. Microscopy and Hi-C analyses showed that the mitotic chromatin organising or "scaffold" proteins are not altered over the region of S. pombe chromatin, but that the amount of chromatin organised around these proteins is diminished. In accordance with the "radial-loop" model of mitotic chromosome structure, we put forward a model whereby the S. pombe chromatin is organised into smaller chromatin loops around a constant organising scaffold. Examination of the histone post-translational modifications over the region of S. pombe chromatin revealed it to be highly heterochromatic, with high levels of H3K9me3 and associated factors such as HP1α and 5meC, and low levels of activating marks. Generation of further mammalian - S. pombe fusion cell lines recapitulated both the distinct mitotic structure and the heterochromatic profile of the inserted S. pombe chromatin. However, insertion of S. pombe DNA into a mouse cell by transfection rather than fusion resulted in a large region of S. pombe DNA that lacked both a distinct structure and heterochromatin. These results suggest that H3K9me3- mediated heterochromatin may influence the structure of chromatin in mitosis, leading to an organisation into smaller chromatin loops than non-heterochromatic regions.