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.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

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.

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.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 3 uppsatser.

No description available.

p13^suc1 from schizosaccharomyces pombe is a member of a family of non-enzymatic cell cycle regulatory proteins called CKS for <strong>C</strong>yclin-<strong>D</strong>ependent kinases <strong>S</strong>ubunit. Other members of this family include CKS1 (S. cerevisiae</em?), CksHsl and CksHs2 (Homo sapiens). The CDKs (CDK1-CDK8) for <strong>C</strong>yclin-<strong>D</strong>ependent <strong>K</strong>inases are a class of Ser/Thr kinases that regulate the cell cycle. The suc1⁺ gene was initially identified as a seppresor of certain CDKl temperature sensitive mutations. Despite the wealth of crystallographic models available plus ample - albeit, sometimes conflicting - evidence from genetics and biochemical studies, an account of the exact physiological role of the CKS proteins remains an elusive goal. In a quest to identify the residues and hence the particular surface region involved in mediating protein-protein interactions with CDK2,1 employed the X-ray structure of Suc1 at 2.7A resolution as guide for a site-specific mutagenesis study. Comparative biochemical and biophysical characterisation of Suc1 and the mutants led to the conclusion that isoleucine-94 and Leucine-96 (located in the hydrophobic patch) are involved in mediating protein-protein interactions with GST-CDK2. This conclusion has since been confirmed by the publication of the X-ray structure of monomeric CksHs1l in a complex with CDK2 by Bourne et al., 1996 (Cell 84: 863-874). An extension of the mutational study to test the hypothesis that Suc1 may utilise conserved residues at the anion-binding site to mediate protein-protein interactions with the Anaphase Promoting Complex (APC) is described. X-ray data has been collected on wild type Suc1 crystals at 100K to 2.3Å resolution. The structure has been resolved and refined to a crystallographic R-factor of 22.6%. S. pombe Suc1 exists as a zinc-stabilised, non strand-exchanged dimer in both the 2.1Å and 2.3Å models. Structural analyses of two Suc1 folding mutants are also presented. The cyclins (A - H) are positive regulatory subunits of CDKs. They share a high degree of homology over a region of about 100 amino acid residues called the "cyclin box". The determination of the crystal structure of cyclin A3 (an N-terminal truncated version of bovine cyclin A) and a CDK2-cyclin A3 complex by other workers has revealed the mechanism of activation of CDKs by cyclins. In S. pombe, the CDKl-cyclin B heterodimeric complex constitutes the mitotic kinase. In order to understand the specificity underlying the CDK-cyclin interaction, I embarked on a structural study of S. pombe cyclin B and CDK1. Both full- length proteins have proven intractable to attempts to overproduce them in a soluble form in E. coli for crystallisation studies. A truncated version of cyclin B (similar to cyclin A3) was designed, cloned and overproduced in E. coli. The cyclin B3 protein was directed into inclusion bodies as insoluble aggregates. Extensive attempts - both in vivo and in vitro - to produce a soluble cyclin B3 proved unsuccessful. Finally, an E. coli co-expression system designed to overproduce CDK1-cyclin A3, CDK1-cyclin B3, CDK1-cyclin B and CDK1-Suc1 complexes is described.

Stuttgart, Univ., Diss., 2005.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 93-103). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Techn. Hochsch., Diss., 2003--Aachen.