Amused: a multi-user software environment diagnostic

As software projects grow in size and complexity, many individuals take over the responsibilities for one project, creating a potential for new errors in the development process. Software version inconsistency, unfamiliarity with the tools used, and software tool restrictions are but some of the problems encountered in a multi-programmer environment. These problems are not always self-evident to the programmer and may require a dedicated software support representative or experienced programmers to assist. These problems can be reduced through the development of a multi-user software environment diagnostic expert system, AMUSED (A Multi-User Software Environment Diagnostic). The AMUSED expert system is designed for use by programmers responsible for creating the executable software releases on a standard copier / duplicator project. Project source code is transported to a common workstation, and linked together with other programmers\u27 code through a linking tool. AMUSED\u27s diagnostic help assists (a) the link process that will be used to create the executable code from the source files, (b) the retrieval of source files from remote sites to the link workstation, and (c) the use of any interfacing connections between the source modules

Studying protein-protein interactions in budding yeast using co-immunoprecipitation

Understanding protein-protein interactions and the architecture of protein complexes in which they work is essential to identify their biological role. Protein co-immunoprecipitation (co-IP) is an invaluable technique used in biochemistry allowing the identification of protein interactors. Here, we describe in detail an immunoaffinity purification protocol as a one step or two-step immunoprecipitation from budding yeast Saccharomyces cerevisiae cells to subsequently detect interactions between proteins involved in the same biological process

Motivation for playing games and motives behind different types of games

Název: Motivace ke hře a motivy u různých typů her Cíle: Cílem této práce je přiblížení principů motivace, vysvětlení motivace člověka ke hře, porovnání a vytvoření přehledu znalostí a teorií o motivaci ke hře a dále vytvoření přehledu rozdílů v motivech u různých typů táborových her. Metody: Jedná se o teoretickou práci s použitím analyticky-syntetické metody. Po provedení analýzy a komparace literární rešerše je syntetizována do stručnější ucelené formy. Dále jsou pomocí dedukce přiřazovány motivy k různým typům her. Výsledky: Zjistili jsme, že motivace je velmi komplexní proces, který se obtížně zkoumá. Odborná veřejnost není ve shodě v otázkách definice, klasifikace, účelu her a motivace k nim, krom toho, že u dětí hra slouží k učení. Akcelerovat poznání o motivaci by mohl poměrně nový obor affektivní neurovědy. Klíčová slova: potřeby, hraní, dělení her, klasifikace her, účel her.Title: Motivation for playing games and motives behind different types of games Objectives: The aim of this work is to give an overview of the principles of motivation, explain motivation for playing games, compare and create a summary of information and theories about motivation for playing games. Then the aim is to create an overview of motives in different types of games. Methods: This is a theoretical work with use of a method of analysis and synthesis. After an analysis and comparison of the literature review it is synthesized into shorter integrated form. Then we use deduction to link underlying motives to different game types. Results: We found that motivation is a very complex process that is difficult to study. The professional public is not in unison in questions of games definition, classification, purpose and motivation for it, except that children's play serves learning. The inquiry of motivation could be accelerated by the fairly new field of affective neuroscience. Keywords: needs, play, game classification, game categorization, game purpose.Sporty v příroděFaculty of Physical Education and SportFakulta tělesné výchovy a sport

Cell polarity protein Spa2 coordinates Chs2 incorporation at the division site in budding yeast

Deposition of additional plasma membrane and cargoes during cytokinesis in eukaryotic cells must be coordinated with actomyosin ring contraction, plasma membrane ingression and extracellular matrix remodelling. The process by which the secretory pathway promotes specific incorporation of key factors into the cytokinetic machinery is poorly understood. Here, we show that cell polarity protein Spa2 interacts with actomyosin ring components during cytokinesis. Spa2 directly binds to cytokinetic factors Cyk3 and Hof1. The lethal effects of deleting the SPA2 gene in the absence of either Cyk3 or Hof1 can be suppressed by expression of the hypermorphic allele of the essential chitin synthase II (Chs2), a transmembrane protein transported on secretory vesicles that makes the primary septum during cytokinesis. Spa2 also interacts directly with the chitin synthase Chs2. Interestingly, artificial incorporation of Chs2 into the cytokinetic machinery allows the localisation of Spa2 at the site of division. In addition, increased Spa2 protein levels promote Chs2 incorporation at the site of division and primary septum formation. Our data indicate that Spa2 is recruited to the cleavage site to co-operate with the secretory vesicle system and particular actomyosin ring components to promote the incorporation of Chs2 into the so-called 'ingression progression complexes' during cytokinesis in budding yeast.ASD was a recipient of a Ramon y Cajal contract (RYC-2010-06156) and received funding from the Cantabria International Campus (http://www.cantabriacampusinternacional.com/Paginas/Cantabria-Campus-de-Excelencia-Internacional.aspx) and via grants BFU2011-23193 and BFU2014-58081-P from the Spanish Ministerio de Economia y Competitividad (co-funded by the European Regional Development Fund) (http:// www.idi.mineco.gob.es/portal/site/MICINN/ menuitem.00d7c011ca2a3753222b7d1001432ea0/?vgnextoid=33881f4368aef110VgnVCM1000001034e20aRCRD) (http://ec.europa.eu/regional_policy/en/funding/erdf/). MF received a Juan de la Cierva contract from the Spanish Ministerio de Economia y Competitividad

PP2A-Cdc55 phosphatase regulates actomyosin ring contraction and septum formation during cytokinesis

Eukaryotic cells divide and separate all their components after chromosome segregation by a process called cytokinesis to complete cell division. Cytokinesis is highly regulated by the recruitment of the components to the division site and through post-translational modifications such as phosphorylations. The budding yeast mitotic kinases Cdc28-Clb2, Cdc5, and Dbf2-Mob1 phosphorylate several cytokinetic proteins contributing to the regulation of cytokinesis. The PP2A-Cdc55 phosphatase regulates mitosis counteracting Cdk1- and Cdc5-dependent phosphorylation. This prompted us to propose that PP2A-Cdc55 could also be counteracting the mitotic kinases during cytokinesis. Here we show that in the absence of Cdc55, AMR contraction and the primary septum formation occur asymmetrically to one side of the bud neck supporting a role for PP2A-Cdc55 in cytokinesis regulation. In addition, by in vivo and in vitro assays, we show that PP2A-Cdc55 dephosphorylates the chitin synthase II (Chs2 in budding yeast) a component of the Ingression Progression Complexes (IPCs) involved in cytokinesis. Interestingly, the non-phosphorylable version of Chs2 rescues the asymmetric AMR contraction and the defective septa formation observed in cdc55 increment mutant cells. Therefore, timely dephosphorylation of the Chs2 by PP2A-Cdc55 is crucial for proper actomyosin ring contraction. These findings reveal a new mechanism of cytokinesis regulation by the PP2A-Cdc55 phosphatase and extend our knowledge of the involvement of multiple phosphatases during cytokinesis

Ingression Progression Complexes Control Extracellular Matrix Remodelling during Cytokinesis in Budding Yeast

Eukaryotic cells must coordinate contraction of the actomyosin ring at the division site together with ingression of the plasma membrane and remodelling of the extracellular matrix (ECM) to support cytokinesis, but the underlying mechanisms are still poorly understood. In eukaryotes, glycosyltransferases that synthesise ECM polysaccharides are emerging as key factors during cytokinesis. The budding yeast chitin synthase Chs2 makes the primary septum, a special layer of the ECM, which is an essential process during cell division. Here we isolated a group of actomyosin ring components that form complexes together with Chs2 at the cleavage site at the end of the cell cycle, which we named ‘ingression progression complexes’ (IPCs). In addition to type II myosin, the IQGAP protein Iqg1 and Chs2, IPCs contain the F-BAR protein Hof1, and the cytokinesis regulators Inn1 and Cyk3. We describe the molecular mechanism by which chitin synthase is activated by direct association of the C2 domain of Inn1, and the transglutaminase-like domain of Cyk3, with the catalytic domain of Chs2. We used an experimental system to find a previously unanticipated role for the C-terminus of Inn1 in preventing the untimely activation of Chs2 at the cleavage site until Cyk3 releases the block on Chs2 activity during late mitosis. These findings support a model for the co-ordinated regulation of cell division in budding yeast, in which IPCs play a central role

Ufd1-Npl4 recruit Cdc48 for disassembly of ubiquitylated CMG helicase at the end of chromosome replication

Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction

Nucleosomes influence multiple steps during replication initiation

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin.American Cancer Society (Postdoctoral Fellowship 123700-PF-13-071-01)National Cancer Institute (U.S.) (Koch Institute Support Grant P30-CA14051

Spt6 is a maintenance factor for centromeric CENP-A

Replication and transcription of genomic DNA requires partial disassembly of nucleosomes to allow progression of polymerases. This presents both an opportunity to remodel the underlying chromatin and a danger of losing epigenetic information. Centromeric transcription is required for stable incorporation of the centromere-specific histone dCENP-A in M/G1 phase, which depends on the eviction of previously deposited H3/H3.3-placeholder nucleosomes. Here we demonstrate that the histone chaperone and transcription elongation factor Spt6 spatially and temporarily coincides with centromeric transcription and prevents the loss of old CENP-A nucleosomes in both Drosophila and human cells. Spt6 binds directly to dCENP-A and dCENP-A mutants carrying phosphomimetic residues alleviate this association. Retention of phosphomimetic dCENP-A mutants is reduced relative to wildtype, while non-phosphorylatable dCENP-A retention is increased and accumulates at the centromere. We conclude that Spt6 acts as a conserved CENP-A maintenance factor that ensures long-term stability of epigenetic centromere identity during transcription-mediated chromatin remodeling