Memoria para optar el título de Bioquímico / La transcripción del DNA, primera etapa de la expresión génica, corresponde a la conversión de un DNA molde a RNA por acción enzimática de la RNA polimerasa (RNAP). En las células de los organismos eucariontes existen tres clases de RNA polimerasas nucleares (RNAP I, II y III). Cada clase de RNAP reconoce promotores específicos mediante la formación de complejos de preiniciación (Pre-initiation Complex o PIC) entre la RNAP y el uso factores de transcripción generales (General Transcription Factors o GTFs), siendo estos últimos los que dan especificidad a cada PIC. Los promotores son secuencias de DNA, normalmente ubicadas río arriba del inicio de la transcripción (Transcription Start Site o TSS), relativamente conservadas en el genoma, que poseen todos los elementos en cis necesarios para el inicio y la regulación de la transcripción de un gen. Los promotores de la RNAP II son los más estudiados y se clasifican en los que poseen caja TATA y en los que no, llamados TATA-Less. Entre los factores de transcripción que se unen a estos promotores están TFIIB, TFIID, TFIIA, entre otros. La caja HomolD es un elemento del promotor mínimo que se encuentra en un promotor de tipo TATA-less de secuencia consenso CAGTCACA, descubierto en genes de proteínas ribosomales de Schizosaccharomyces pombe, y que es capaz de reclutar la RNAP II e iniciar la transcripción. Estudios recientes indican que para iniciar la transcripción desde estos promotores se necesitan los siguientes GTFs: TFIID, TFIIB, TFIIF, TFIIH y TFIIE. Además, se ha propuesto que el factor Rrn7 (antes conocido como miembro de la maquinaria de la RNAP I) podría interaccionar con algunos de estos GTFs, como por ejemplo TFIIB y/o TBP (miembro del complejo TFIID), lo que le conferiría a Rrn7 la facultad de unirse a la caja HomolD y dirigir la transcripción. Sin embargo, aún se desconoce cuáles de estos factores junto a Rrn7 conforman el PIC, que se ensambla sobre la caja HomolD. En este trabajo se estudió la participación de los factores TFIIB y TBP, como miembros candidatos del complejo de pre-iniciación formado sobre la caja HomolD. Mediante ensayos en geles de retardo o EMSA y ensayos de inmunoprecipitación de cromatina se analizaron las interacciones DNA-proteína y proteína-proteína. Para realizar los ensayos de EMSA fue necesario expresar y purificar la proteína Rrn7 recombinante utilizando una etiqueta de histidina, la que se utilizó junto a TBP y TFIIB. Además, se purificaron anticuerpos policlonales contra ambos factores, y fueron usados en ensayos de EMSA a modo de control. Los resultados de los EMSA muestran una intensificación de la banda representativa al complejo Rrn7-caja HomolD al agregar cada factor a la reacción (TFIIB o TBP). Los ensayos de ChIP se realizaron en una cepa silvestre de S. pombe utilizando los mismos anticuerpos purificados contra los factores TBP y TFIIB. El DNA obtenido, fue sometido a amplificación mediante PCR utilizando partidores específicos para la caja HomolD. Además se realizaron dos controles, uno utilizando partidores que amplificaron parte del promotor y del primer exón del gen nmt1 para la caja TATA como control positivo, y otros para una región del gen de actina (act1) como control negativo. Se observó amplificación del DNA representativo a la caja HomolD, al precipitar cada factor, reflejando la presencia de éstos en la región promotora. Los resultados obtenidos en este trabajo muestran in vitro (EMSA) un efecto potenciador de TBP y TFIIB sobre la formación del complejo Rrn7-caja HomolD, lo que es complementario a la presencia mostrada in vivo (ChIP) de estos factores en el promotor. De estos experimentos se concluye la participación de ambos factores en la formación del complejo de pre-iniciación sobre la caja HomolD. Además los resultados de EMSA indicaron que TFIIB posee un efecto potenciador mayor que TBP a una determinada concentración. Por otro lado, en ensayos EMSA ambos factores produjeron un desplazamiento o retardo de la banda del complejo Rrn7-HomoID, pero el desplazamiento provocado por TBP fue más intenso y estable, a diferencia del de TFIIB que fue solo temporal. Estos resultados sugieren que los roles que cumplen los factores TFIIB y TBP en el PIC en la caja HomolD, son el de un factor reclutador de Rrn7 al promotor y el de un factor potenciador de la interacción proteína-DNA, respectivamente / DNA transcription, the first stage of gene expression, is defined as the conversion of a template DNA to RNA by the enzymatic action of the RNA polymerase (RNAP). In the eukaryotic cells there are three classes of nuclear polymerases (RNAP I, II and III). Each class of RNAP recognizes specific promoters through the formation of pre initiation complexes (PIC) between the polymerase and general transcription factors (GTF). The latter provide the specificity to each PIC. These promoters are relatively conserved DNA sequences that usually are located upstream from the transcriptional start site (TSS), and have all the cis elements needed for the start and regulation of the transcription of a gene. RNAP II promoters are the most studied ones, and are categorized as those that have a TATA box or those that lack a TATA box, called TATA-less promoters. The factors that bind to these promoters are TFIIB, TFIIA and TFIID, among others. The HomolD box is a core promoter element (CPE) that is found in a TATAless type promoter, whose consensus sequence is CAGTCACA. This CPE was discovered in ribosomal protein genes of the fission yeast Schizosaccharomyces pombe, and recruits RNAP II and starts transcription. Recent studies have shown that to start transcription from these RNAP II promoters the GTFs: TFIID, TFIIB, TFIIF, TFIIH and TFIIE are needed. Furthermore, it has been proposed that the Rrn7 factor (formerly known as a member of the RNAP I machinery) interacts with some of these GTFs, for instance with TFIIB and/or TBP (member of the TFIID complex), which could confer Rrn7 the ability to bind the HomolD box and promote transcription. However, it is still unknown which of these factors in addition to Rrn7 conform the PIC that assembles over the HomolD box. In this work the participation of the factors TBP and TFIIB, as candidate members of the PIC formed over the HomolD box, was studied. Using Electrophoretic Mobility Shift Assays (EMSA) and Chromatin Immunoprecipitation assays (ChIP), DNA-protein and protein-protein interactions were analyzed. To carry out the EMSA studies, it was necessary to express and purify the recombinant Rrn7 protein using a 6xHis tag, which was then used with the factors TBP and TFIIB, also recombinant from S. pombe. Additionally, polyclonal antibodies for both factors were purified and used in EMSA experiments as a control. The EMSA results showed an intensification of the band representative of the complex when each factor was added to the reaction (TBP or TFIIB). ChIP assays were performed in a wild type S. pombe strain, using the same antibodies purified for TFIIB and TBP. The DNA obtained was amplified with PCR using specific primers for the HomolD box. Two controls were carried out, one using primers that amplified part of the promoter and the first exon of the nmt1 gene for the TATA box as a positive control. The other control used primers for a region of the actin gene (act1) as a negative control. When each factor was precipitated, an amplification of the HomolD box sequence, revealing the presence of both factors at the promoter region. Results obtained in this work show in vitro (EMSA) an enhancer effect of TBP and TFIIB on the formation of the Rrn7-HomolD box complex, which is complementary to the presence shown in vivo (ChIP) of these factors in the promoter. From these experiments it is concluded that both factors participate in the formation of the PIC over the HomolD box. Furthermore, the EMSA results indicate that TFIIB has a greater enhancer effect than TBP. On the other side, EMSA assays of both factors produced a shift of the complex Rrn7-HomolD band, but the one produced by TBP was more intense and stable, in comparison with the complex produced by TFIIB, which was temporal and less intense. These results suggest that TFIIB recruits Rrn7 to the PIC and that TBP enhances the interaction between protein and DNA of the PIC at the HomolD box

No description available.

Cid1 is a cytoplasmic member of a novel class of regulatory poly(A) polymerases discovered recently in yeast, worms and vertebrates. Previous genetic studies in the fission yeast, Schizosaccharomyces pombe, suggested a role for Cid1 in the checkpoint response to replication stress, but it was not known how a poly(A) polymerase might contribute to this response. Further investigations into the mode of action of Cid1 were therefore undertaken in this study. Cid1 is likely to target specific RNAs for polyadenylation; potential RNA substrates were identified using the complementary methods of microarray hybridisation and whole proteome analysis using two-dimensional liquid chromatography. These experiments revealed that Cid1 does not affect RNAs during normal, unperturbed growth but instead alters the expression of specific subsets of genes during replication stress. Many RNAs affected by Cid1 in these circumstances were cell-cycle dependent and telomeric transcripts, including those encoding histones and a novel RecQ helicase, Rqh2. As Cid1 lacks an RNA recognition motif, it is unlikely to bind selectively to RNA targets on its own. Cid1-interacting proteins were identified using yeast two-hybrid and tandem affinity purification methods. From these studies, novel members of a Cid1 complex have been discovered including: a previously uncharacterised metallo-beta-lactamase, RNA-binding proteins, ribosomal proteins and a telomere-binding protein. Together, these approaches are leading to a model for the role of cytoplasmic polyadenylation by Cid1 in checkpoint control.

The Schizosaccharomyces pombe cdc21⁺ gene product is related to the Mcm2-3-5 family of replication proteins. By phylogeny analysis of their protein sequences and screening for cdc21⁺-related sequences using molecular probes I have suggested that at least six types of cdc21⁺-related genes may be present in the yeast genome. The isolation of interaction suppressors of the cdc21^ts mutant was attempted by overexpression of an S. pombe cDNA library. Two cDNAs were isolated, ts11⁺ and dom1⁺, whose overexpression specifically affected the viability of cdc21^ts cells under certain conditions. The predicted dom1 protein is 60% identical to the budding yeast HMG-like Nhp2 protein. I have studied the phenotype of S. pombe cells overexpressing the cdc21⁺ gene and amino-terminal truncations of it. Overexpression of the cdc21⁺ gene caused cell elongation but cells were not significantly affected in growth rate. Cells overexpressing the carboxyl-terminal part of cdc21⁺ arrested in S phase and also entered mitosis in the absence of nuclear division. The possibility that chromosomes in cdc21^ts arrested cells may be damaged was investigated by pulsed field gel electrophoresis. No differences could be found compared to wild-type chromosomes. I have also studied the arrest phenotype of cdc21 rad1 and cdc21 cdc2.3w double mutants. Both strains entered mitosis at the restrictive temperature indicating that cdc21^ts cells arrest in S phase and may contain DNA damage. I have generated two new mutant alleles of cdc21⁺. The first allele was made by deleting most of the cdc21⁺ open reading frame (ORF). The second allele was constructed by placing the cdc21⁺ ORF under control a regulatable promoter. The resulting construct was used to complement the cdc21 deletion. Both mutants were inviable under appropriate conditions arresting in S phase as elongated cells, although a proportion of them (15-20%) entered mitosis in the absence of nuclear division.

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.

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.