Search

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4 based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.

An optimised extraction protocol for the analysis of Saccharomyces cerevisiae aqueous and organic metabolites by nuclear magnetic resonance spectroscopy that allows the identification and quantification of up to 50 different compounds is presented. The method was compared with other metabolic profiling protocols for S. cerevisiae, where generally different analytical techniques are applied for metabolite quantification. In addition, the analysis of intact S. cerevisiae cells by HRMAS was implemented for the first time as a complementary method. The optimised protocols were applied to study the metabolic effect of glucose and galactose on S. cerevisiae growth. Furthermore, the metabolic reaction of S. cerevisiae to osmotic stress has been studied.

Fission yeast cells can be synchronized by cell cycle arrest and release or by size selection. Cell cycle arrest synchronization is based on the block and release of temperature-sensitive cell cycle mutants or treatment with drugs. The most widely used approaches are cdc10-129 for G1; hydroxyurea (HU) for early S-phase; cdc25-22 for G2, and nda3-KM311 for mitosis. Cells can also be synchronized by size selection using centrifugal elutriation or a lactose gradient. Here we describe the methods most commonly used to synchronize fission yeast cells.

Memoria realizada por la licenciada Dª Marta Tormos Pérez en el Centro Mixto Instituto de Biología Funcional y Genómica, CSIC/Universidad de Salamanca para optar al grado de Doctora en Biotecnología., En este estudio se ha analizado la función de dos proteínas de plegamiento tipo histona que interaccionan físicamente entre sí formando complejos implicados en dos procesos de gran relevancia fisiológica como son la segregación y la reparación del DNA. Estas proteínas fueron identificadas inicialmente en células de mamíferos como componentes constitutivos de la región interna del cinetocoro y se las denominó CENP-S y CENP-X (centromeric protein). En trabajos posteriores, realizados también en células de mamíferos, se identificaron formando un complejo con la DNA helicasa FANCM que participa en la ruta de reparación de la Anemia de Fanconi, denominándose MHF1 y MHF2 (FANCM- associated histone-fold protein). Así pues en eucariotas superiores hablamos de estas proteínas como CENP-S y -X si tratamos sobre la segregación del material genético o como MHF1 y 2, respectivamente, si nos referimos a su actividad en la reparación del DNA. La estructura de estas proteínas, su interacción física y su implicación en los procesos mencionados están altamente conservados desde humanos hasta levaduras indicando su importancia fisiológica. Sin embargo, muchos datos acerca de su regulación y actividad son aún desconocidos. Debido a su alto grado de conservación, su estudio en eucariotas unicelulares como las levaduras facilitará la disección y el entendimiento de su función.

14 páginas, 5 figuras, 1 tabla., The LRRK2 gene (Leucine-Rich Repeat Kinase 2, PARK8) is mutated in a significant number of cases of autosomal dominant Parkinson's disease (PD) and in some sporadic cases of late-onset PD. LRRK2 is a large, complex protein that comprises several interaction domains: armadillo, ankyrin, leucine-rich repeats and WD40 domains; two catalytic domains: ROC-GTPase and serine/threonine kinase; and a COR domain (unknown function). Pathogenic mutations are scattered all over the domains of LRRK2, although the prevalence of mutations in some domains is higher (ROC-GTPase, COR and kinase). In this work, we model the structure of each domain to predict and explore the effects of described missense mutations and polymorphisms. The results allow us to postulate the possible effects of pathogenic mutations in the function of the protein, and hypothesize the importance of some polymorphisms that have not been linked directly to PD, but act as risk factors for the disease. In our analysis, we also study the effects of PD-related mutations in the kinase domain structure and in the phosphorylation of the activation loop to determine effects on kinase activity., Contract Grant sponsor: The Ministerio de Ciencia e Innovación, Spain; Contract Grant number: SAF2009-10434; Contract Grant sponsor: Generalitat Valenciana; Contract Grant number: ACOMP/2010/096.