RESUMEN
The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.
Asunto(s)
Proteínas 14-3-3 , Segregación Cromosómica , Cinetocoros , Mitosis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cinetocoros/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Microtúbulos/metabolismo , Cromosomas Fúngicos/metabolismo , Cromosomas Fúngicos/genéticaRESUMEN
Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF-type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and interaction analyses. We describe a Rep-STB bypass system in which a plasmid engineered to non-covalently associate with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animales , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina/genética , Cromosomas/metabolismo , Plásmidos/genética , Cromatina/genética , Cromatina/metabolismo , Mamíferos/genéticaRESUMEN
To understand the physical phenomena responsible for radiation damage of the materials used in nuclear reactors, and thus study their operation life and/or efficiency, it is required to simulate the conditions by exposing the materials to energetic ions. Ceria (CeO2) has been proposed as one of the inert matrices for the transmutation of minor actinides in the futuristic inert matrix fuel (IMF) concept. The inert matrix should also contain burnable poison to compensate for the initial reactivity of fuel. In this context, gadolinium (Gd) is an excellent burnable poison with a high neutron absorption cross-section. In view of this, Gd2O3-CeO2 nano-powders were synthesized and sintered at 800 °C and 1300 °C to obtain different grain sizes and morphologies. FESEM and TEM were carried out to study the grain size of pristine pellets. The sintered pellets were irradiated with 80-MeV Ag ions (electronic energy loss (Se) regime) at room temperature to emulate the effect of fission fragments. For analysis of the effect of grain size on the irradiation-induced structural degradation at different fluences, GIXRD and Raman spectroscopy were performed. Significantly large damage has been observed for the smaller grain-sized samples (sintered at 800 °C) as compared to the large grain-sized sample (sintered at 1300 °C). Neither of the samples amorphized under the present experimental conditions as indicated by the presence of the Raman-active T2g mode (centred at 462 cm-1) and all the XRD peaks of fluorite cubic structure up to the highest fluence employed (1 × 1014 ions cm-2). X-ray photoelectron spectroscopy results demonstrate that Ce4+ to Ce3+ and vacancy-related isolated clusters are the main defects produced in the systems. The radiation tolerance behaviour of the samples is understood with the help of thermal spike simulation, which indicates higher transient lattice temperatures with longer duration in the smaller grain-sized sample upon irradiation. Gd-doped ceria thus possesses good radiation stability in the Se regime, indicating its potential for application in IMFs.
RESUMEN
Minichromosome maintenance (Mcm) proteins are well-known for their functions in DNA replication. However, their roles in chromosome segregation are yet to be reviewed in detail. Following the discovery in 1984, a group of Mcm proteins, known as the ARS-nonspecific group consisting of Mcm13, Mcm16-19, and Mcm21-22, were characterized as bonafide kinetochore proteins and were shown to play significant roles in the kinetochore assembly and high-fidelity chromosome segregation. This review focuses on the structure, function, and evolution of this group of Mcm proteins. Our in silico analysis of the physical interactors of these proteins reveals that they share non-overlapping functions despite being copurified in biochemically stable complexes. We have discussed the contrasting results reported in the literature and experimental strategies to address them. Taken together, this review focuses on the structure-function of the ARS-nonspecific Mcm proteins and their evolutionary flexibility to maintain genome stability in various organisms.
Asunto(s)
Segregación Cromosómica , Eucariontes , Proteínas de Ciclo Celular/genética , Eucariontes/genética , Cinetocoros , Proteínas de Mantenimiento de Minicromosoma/genéticaRESUMEN
Equipartitioning by chromosome association and copy number correction by DNA amplification are at the heart of the evolutionary success of the selfish yeast 2-micron plasmid. The present analysis reveals frequent plasmid presence near telomeres (TELs) and centromeres (CENs) in mitotic cells, with a preference towards the former. Inactivation of Cdc14 causes plasmid missegregation, which is correlated to the non-disjunction of TELs (and of rDNA) under this condition. Induced missegregation of chromosome XII, one of the largest yeast chromosomes which harbors the rDNA array and is highly dependent on the condensin complex for proper disjunction, increases 2-micron plasmid missegregation. This is not the case when chromosome III, one of the smallest chromosomes, is forced to missegregate. Plasmid stability decreases when the condensin subunit Brn1 is inactivated. Brn1 is recruited to the plasmid partitioning locus (STB) with the assistance of the plasmid-coded partitioning proteins Rep1 and Rep2. Furthermore, in a dihybrid assay, Brn1 interacts with Rep1-Rep2. Taken together, these findings support a role for condensin and/or condensed chromatin in 2-micron plasmid propagation. They suggest that condensed chromosome loci are among favored sites utilized by the plasmid for its chromosome-associated segregation. By homing to condensed/quiescent chromosome locales, and not over-perturbing genome homeostasis, the plasmid may minimize fitness conflicts with its host. Analogous persistence strategies may be utilized by other extrachromosomal selfish genomes, for example, episomes of mammalian viruses that hitchhike on host chromosomes for their stable maintenance.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Unión al ADN/genética , Complejos Multiproteicos/genética , Plásmidos/genética , Saccharomycetales/genética , Adenosina Trifosfatasas/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , División Celular , Centrómero/metabolismo , Segregación Cromosómica/genética , Cromosomas/genética , Replicación del ADN/genética , ADN de Hongos/genética , Proteínas de Unión al ADN/metabolismo , Heterocromatina/metabolismo , Complejos Multiproteicos/metabolismo , Plásmidos/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/metabolismo , Telómero/metabolismo , Transactivadores/genéticaRESUMEN
Despite recent advancements in the development of catalytic asymmetric electrophile induced lactonization reactions of olefinic carboxylic acids, the archetypical hydrolactonization has long remained an unsolved and well-recognized challenge. Here, we report the realization of a catalytic asymmetric hydrolactonization using a confined imidodiphosphorimidate (IDPi) Brønsted acid catalyst. The method is operationally simple, scalable, and compatible with a wide variety of substrates. Its potential is showcased with concise syntheses of the sesquiterpenes (-)-boivinianin A and (+)-gossonorol. Through in-depth physicochemical and DFT analyses, we derive a nuanced picture of the mechanism and enantioselectivity of this reaction.
RESUMEN
Regulation of gene expression programs is crucial for the survival of microbial pathogens in host environments and for their ability to cause disease. Here we investigated the epigenetic regulator RSC (Remodels the Structure of Chromatin) in the most prevalent human fungal pathogen Candida albicans. Biochemical analysis showed that CaRSC comprises 13 subunits and contains two novel non-essential members, which we named Nri1 and Nri2 (Novel RSC Interactors) that are exclusive to the CTG clade of Saccharomycotina. Genetic analysis showed distinct essentiality of C. albicans RSC subunits compared to model fungal species suggesting functional and structural divergence of RSC functions in this fungal pathogen. Transcriptomic and proteomic profiling of a conditional mutant of the essential catalytic subunit gene STH1 demonstrated global roles of RSC in C. albicans biology, with the majority of growth-related processes affected, as well as mis-regulation of genes involved in morphotype switching, host-pathogen interaction and adaptive fitness. We further assessed the functions of non-essential CaRSC subunits, showing that the novel subunit Nri1 and the bromodomain subunit Rsc4 play roles in filamentation and stress responses; and also interacted at the genetic level to regulate cell viability. Consistent with these roles, Rsc4 is required for full virulence of C. albicans in the murine model of systemic infection. Taken together, our data builds the first comprehensive study of the composition and roles of RSC in C. albicans, showing both conserved and distinct features compared to model fungal systems. The study illuminates how C. albicans uses RSC-dependent transcriptional regulation to respond to environmental signals and drive survival fitness and virulence in mammals.
Asunto(s)
Candida albicans/genética , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiología , Candida albicans/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/genética , Proteómica/métodos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Virulencia/genéticaRESUMEN
We disclose a general catalytic enantioselective Diels-Alder reaction of exo-enones with dienes to give spirocyclanes. The obtained products feature highly congested quaternary stereogenic spirocenters and are used in concise total and formal syntheses of several sesquiterpenes, including of α-chamigrene, ß-chamigrene, laurencenone C, colletoic acid, and omphalic acid. The stereo- and regioselectivities of our spirocyclizing cycloaddition are effectively controlled by strongly acidic and confined imidodiphosphorimidate catalysts. Computational studies shed light on the origin of reactivity and selectivity.
Asunto(s)
Sesquiterpenos , Reacción de Cicloadición , EstereoisomerismoRESUMEN
Shugoshin proteins are evolutionarily conserved across eukaryotes, with some species-specific cellular functions, ensuring the fidelity of chromosome segregation. They act as adaptors at various subcellular locales to mediate several protein-protein interactions in a spatio-temporal manner. Here, we characterize shugoshin (Sgo1) in the human fungal pathogen Candida albicans. We observe that Sgo1 retains its centromeric localization and performs its conserved functions of regulating the sister chromatid biorientation, centromeric condensin localization, and maintenance of chromosomal passenger complex (CPC). We identify novel roles of Sgo1 as a spindle assembly checkpoint (SAC) component with functions in maintaining a prolonged SAC response by retaining Mad2 and Bub1 at the kinetochores in response to improper kinetochore-microtubule attachments. Strikingly, we discover the in vivo localization of Sgo1 along the length of the mitotic spindle. Our results indicate that Sgo1 performs a hitherto unknown function of facilitating timely disassembly of the mitotic spindle in C. albicans. To summarize, this study unravels a unique functional adaptation of shugoshin in maintaining genomic stability.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Candida albicans/fisiología , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Candidiasis/microbiología , Cromátides/metabolismo , Segregación Cromosómica , Proteínas Fúngicas/metabolismo , Inestabilidad Genómica , Humanos , Cinetocoros/metabolismo , MitosisRESUMEN
Fungal pathogens constantly sense and respond to the environment they inhabit, and this interaction is vital for their survival inside hosts and exhibiting pathogenic traits. Since such responses often entail specific patterns of gene expression, regulators of chromatin structure contribute to the fitness and virulence of the pathogens by modulating DNA accessibility to the transcriptional machinery. Recent studies in several human and plant fungal pathogens have uncovered the SWI/SNF group of chromatin remodelers as an important determinant of pathogenic traits and provided insights into their mechanism of function. Here, we review these studies and highlight the differential functions of these remodeling complexes and their subunits in regulating fungal fitness and pathogenicity. As an extension of our previous study, we also show that loss of specific RSC subunits can predispose the human fungal pathogen Candida albicans cells to filamentous growth in a context-dependent manner. Finally, we consider the potential of targeting the fungal SWI/SNF remodeling complexes for antifungal interventions.
Asunto(s)
Proteínas de Unión al ADN , Factores de Transcripción , Candida albicans/genética , Candida albicans/metabolismo , Cromatina , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Humanos , Factores de Transcripción/metabolismoRESUMEN
Candida albicans, the most prevalent fungal pathogen, exists as a commensal in the human host. It is subjected to myriad physiological stress conditions in different host niches, which jeopardizes its fitness to survive and propagate as an established commensal. C. albicans has highly labile chromatin which gets remodeled in response to the stress conditions to facilitate the expression of several stress-responsive genes. Several epigenetic factors including histone variants, histone modifiers and chromatin remodelers that define the chromatin architecture play crucial roles in the regulation of the stress-responsive genes in this organism. Here we investigated the roles of the ATP-dependent chromatin remodeler RSC (Remodel the Structure of Chromatin) in several stress responses in C. albicans, by targeting the key ATPase component, Sth1, given its profound and similar roles exist in Saccharomyces cerevisiae. We have unraveled the crucial roles of the RSC complex (Sth1) in maintaining cell wall integrity and fighting against osmotic and oxidative stresses. We found that the mutant conditionally depleted of Sth1 was sensitive to the cell wall disrupting agents, and the mutant without exposure to any stressor accumulated higher chitin content in the cell wall as a defense mechanism to restore the cell wall integrity. Further, this was supported by the phosphorylation of MAPK1 protein Mkc1, which happens due to activation of the cell wall integrity pathway PKC1. We also observed the Sth1 mutant to be sensitive to oxidative and osmotic stresses in vitro, which are very important and imparted by the host defense mechanism. This suggests that the mutant could get attenuated and hence become less virulent than the wild-type when loss of function of Sth1 happens. We also found that Sth1 has a crucial role in maintaining genomic integrity as sth1 mutant cells accumulate extensive DNA damages and show the loss in cell viability. Overall this work suggests that Sth1 has an important role in fighting against some of the clinically relevant and physiologically important stresses. It also has a crucial role in fighting against stress to the genomic integrity and hence functions in DNA damage repair.
Asunto(s)
Candida albicans , Cromatina , Daño del ADN , Proteínas Fúngicas , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Candida albicans/enzimología , Candida albicans/genética , Proteínas de Ciclo Celular/química , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histonas/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A copper-mediated coupling reaction between ynamides and diazo-compounds to produce N-allenamides is reported for the first time. This method enables facile and rapid access to terminal N-allenamides by using commercially available TMS-diazomethane with wide functional group compatibility on the nitrogen. Furthermore, the ubiquity of molecules containing a fluorine moiety in medicine, in agricultural, and material science requires the continuous search of new building blocks, including this unique surrogate. The CuI/diazo protocol was successfully applied to the synthesis of fluorine-substituted N-allenamides. DFT calculations provided insights in the mechanism involved.
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Cobre , Flúor , Compuestos Azo , CatálisisRESUMEN
A higher order organization of the centromeres in the form of clustering of these DNA loci has been observed in many organisms. While centromere clustering is biologically significant to achieve faithful chromosome segregation, the underlying molecular mechanism is yet to be fully understood. In budding yeast, a kinetochore-associated protein Slk19 is shown to have a role in clustering in association with the microtubules whereas removal of either Slk19 or microtubules alone does not have any effect on the centromere clustering. Furthermore, Slk19 is non-essential for growth and becomes cleaved during anaphase whereas clustering being an essential event occurs throughout the cell cycle. Hence, we searched for an additional factor involved in the clustering and since the integrity of the kinetochore complex is shown to be crucial for centromere clustering, we restricted our search within the complex. We observed that the outermost kinetochore protein Dam1 promotes centromere clustering through stabilization of the kinetochore integrity. While in the absence of Dam1 we failed to detect Slk19 at the centromere, on the other hand, we found almost no Dam1 at the centromere in the absence of Slk19 and microtubules suggesting interdependency between these two pathways. Strikingly, we observed that overexpression of Dam1 or Slk19 could restore the centromere clustering largely in the cells devoid of Slk19 and microtubules or Dam1, respectively. Thus, we propose that in budding yeast, centromere clustering is achieved at least by two parallel pathways, through Dam1 and another via Slk19, in concert with the microtubules suggesting that having a dual mechanism may be crucial for ensuring microtubule capture by the point centromeres where each attaches to only one microtubule.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrómero/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , División Celular , Centrómero/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Unión Proteica , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We describe a highly enantioselective Diels-Alder reaction of cross-conjugated cyclohexadienones with cyclopentadiene, in which five stereocenters are effectively controlled by a strongly acidic and confined imidodiphosphorimidate catalyst. Our approach provides tricyclic products in excellent stereoselectivity. We also report methods to convert the obtained products into useful intermediates and a computational study that aids in gaining deeper insight into the reaction mechanism and origin of stereoselectivity.
RESUMEN
The histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly.
Asunto(s)
Proteínas Cromosómicas no Histona/química , Proteínas de Unión al ADN/química , Histonas/química , Simulación de Dinámica Molecular , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histonas/genética , Histonas/metabolismo , Dominios Proteicos , Estabilidad Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SolubilidadRESUMEN
Direct enantioselective α-alkylation of 2-alkylpyridines provides access to chiral pyridines via an operationally simple protocol that obviates the need for prefunctionalization or preactivation of the substrate. The alkylation is accomplished using chiral lithium amides as noncovalent stereodirecting auxiliaries. Crystallographic and solution NMR studies provide insight into the structure of well-defined chiral aggregates in which a lithium amide reagent directs asymmetric alkylation.
Asunto(s)
Litio/química , Compuestos Organometálicos/química , Piridinas/química , Piridinas/síntesis química , Alquilación , Estructura Molecular , EstereoisomerismoRESUMEN
An underlying theme in the segregation of low-copy bacterial plasmids is the assembly of a 'segrosome' by DNA-protein and protein-protein interactions, followed by energy-driven directed movement. Analogous partitioning mechanisms drive the segregation of host chromosomes as well. Eukaryotic extra-chromosomal elements, exemplified by budding yeast plasmids and episomes of certain mammalian viruses, harbor partitioning systems that promote their physical association with chromosomes. In doing so, they indirectly take advantage of the spindle force that directs chromosome movement to opposite cell poles. Molecular-genetic, biochemical and cell biological studies have revealed several unsuspected aspects of 'chromosome hitchhiking' by the yeast 2-micron plasmid, including the ability of plasmid sisters to associate symmetrically with sister chromatids. As a result, the plasmid overcomes the 'mother bias' experienced by plasmids lacking a partitioning system, and elevates itself to near chromosome status in equal segregation. Chromosome association for stable propagation, without direct energy expenditure, may also be utilized by a small minority of bacterial plasmids-at least one case has been reported. Given the near perfect accuracy of chromosome segregation, it is not surprising that elements residing in evolutionarily distant host organisms have converged upon the common strategy of gaining passage to daughter cells as passengers on chromosomes.
Asunto(s)
Cromosomas Fúngicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Segregación Cromosómica/genética , Replicación del ADN/genética , Plásmidos/genética , Saccharomyces cerevisiae/genéticaRESUMEN
An unprecedented direct C-C coupling reaction of unprotected primary amines with active methylene compounds is reported. The reaction involves a biomimetic deamination of amines which was achieved under conditions free of metallic reagents and strong oxidizing agents. A wide range of primary amines was reacted with different active methylene compounds to provide structurally diverse trisubstituted alkenes and dihydropyridines. A kinetic study revealed an activation barrier of 10.1 kcal mol-1 for the conversion of a key intermediate of the reaction.
RESUMEN
We report here the stabilization of the cubic phase under ambient conditions in the thin films of zirconia synthesized by electron beam evaporation. The cubic phase stabilization was achieved without the use of chemical stabilizers and/or concurrent ion beam bombardment. Films of two different thicknesses (660 nm and 140 nm) were deposited. While the 660 nm as-deposited films were in the cubic phase, as indicated by X-ray diffraction and Raman spectroscopy, the 140 nm as-deposited films were amorphous and the transformation to the cubic phase was obtained after thermal annealing. Extended X-ray absorption fine structure measurements revealed the existence of oxygen vacancies in the local structure surrounding zirconium for all films. However, the amount of these oxygen vacancies was found to be significantly higher for the amorphous films as compared to that for the films in the cubic phase (660 nm as-deposited and 140 nm annealed films). The stabilization of the cubic phase is attributed to the breaking of the oxygen-zirconium bonds due to the presence of the oxygen vacancies, which results in the suppression of the soft X2- mode of vibration of the oxygen sub-lattice. Our first-principles modeling under the framework of density functional theory shows that the cubic structure with oxygen vacancies is indeed more stable under ambient conditions than its pristine (without vacancies) counterpart due to breaking of the oxygen bonds. The requirement of a critical amount of these vacancies for cubic phase stabilization is discussed.
RESUMEN
The 2-µm plasmid of the budding yeast Saccharomyces cerevisiae achieves a high chromosome-like stability with the help of four plasmid-encoded (Rep1, Rep2, Raf1 and Flp) and several host-encoded proteins. Rep1 and Rep2 and the DNA locus STB form the partitioning system ensuring equal segregation of the plasmid. The Flp recombinase and its target sites FRTs form the amplification system which is responsible for the steady state plasmid copy number. In this work we show that the absence of Raf1 can affect both the plasmid stability and the steady sate copy number. We also show that the Rep proteins do bind to the promoter regions of the 2-µm encoded genes, as predicted by earlier models and Raf1 indeed blocks the formation of the Rep1-Rep2 repressor complex not by blocking the transcription of the REP1 and REP2 genes but by physically associating with the Rep proteins and negating their interactions. This explains the role of Raf1 in both the partitioning and the amplification systems as the Rep1-Rep2 complex is believed to modulate both these systems. Based on this study, we have provided, from a systems biology perspective, a model for the mechanism of the 2-µm plasmid maintenance.