Identification of a novel type of spacer element required for imprinting in fission yeast

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during laggingstrand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fullyprocessed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers

Repeated evolution of self-compatibility for reproductive assurance

Sexual reproduction in eukaryotes requires the fusion of two compatible gametes of opposite sexes or mating types. To meet the challenge of finding a mating partner with compatible gametes evolutionary mechanisms such as hermaphroditism and self-fertilisation have repeatedly evolved. Combining insight from comparative genomics, computer simulations and experimental evolution in fission yeast, we shed light on the conditions promoting separate mating types or self-compatibility by mating-type switching. Analogous to multiple independent transitions between switchers and non-switchers in natural populations mediated by structural genomic changes, novel switching genotypes were readily evolving under selection in experimental populations. Detailed fitness measurements accompanied by computer simulations show the benefits and costs of switching during sexual and asexual reproduction governing the occurrence of both strategies in nature. Our findings illuminate the trade-off between the benefits of reproductive assurance and its fitness costs under benign conditions governing the evolution of self-compatibility

The role of the RACK1 ortholog Cpc2p in modulating pheromone-induced cell cycle arrest in fission yeast

The detection and amplification of extracellular signals requires the involvement of multiple protein components. In mammalian cells the receptor of activated C kinase (RACK1) is an important scaffolding protein for signal transduction networks. Further, it also performs a critical function in regulating the cell cycle by modulating the G1/S transition. Many eukaryotic cells express RACK1 orthologs, with one example being Cpc2p in the fission yeast Schizosaccharomyces pombe. In contrast to RACK1, Cpc2p has been described to positively regulate, at the ribosomal level, cells entry into M phase. In addition, Cpc2p controls the stress response pathways through an interaction with Msa2p, and sexual development by modulating Ran1p/Pat1p. Here we describe investigations into the role, which Cpc2p performs in controlling the G protein-mediated mating response pathway. Despite structural similarity to Gβ-like subunits, Cpc2p appears not to function at the G protein level. However, upon pheromone stimulation, cells overexpressing Cpc2p display substantial cell morphology defects, disorientation of septum formation and a significantly protracted G1 arrest. Cpc2p has the potential to function at multiple positions within the pheromone response pathway. We provide a mechanistic interpretation of this novel data by linking Cpc2p function, during the mating response, with its previous described interactions with Ran1p/Pat1p. We suggest that overexpressing Cpc2p prolongs the stimulated state of pheromone-induced cells by increasing ste11 gene expression. These data indicate that Cpc2p regulates the pheromone-induced cell cycle arrest in fission yeast by delaying cells entry into S phase

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

Computational modelling of meiotic entry and commitment

In response to developmental and environmental conditions, cells exit the mitotic cell cycle and enter the meiosis program to generate haploid gametes from diploid germ cells. Once cells decide to enter the meiosis program they become irreversibly committed to the completion of meiosis irrespective of the presence of cue signals. How meiotic entry and commitment occur due to the dynamics of the regulatory network is not well understood. Therefore, we constructed a mathematical model of the regulatory network that controls the transition from mitosis to meiosis in Schizosaccharomyces pombe. Upon nitrogen starvation, yeast cells exit mitosis and undergo conjugation and meiotic entry. The model includes the regulation of Mei2, an RNA binding protein required for conjugation and meiotic entry, by multiple feedback loops involving Pat1, a kinase that keeps cells in mitosis, and Ste11, a transcription activator required for the sexual differentiation. The model accounts for various experimental observations and demonstrates that the activation of Mei2 is bistable, which ensures the irreversible commitment to meiosis. Further, we show by integrating the meiosis-specific regulation with a cell cycle model, the dynamics of cell cycle exit, G1 arrest and entry into meiosis under nitrogen starvation. © 2017 The Author(s)

The origins of the Selden map of China: scientific analysis of the painting materials and techniques using a holistic approach

Since the 'rediscovery' of the Selden map of China, an early seventeenth century map of Asia, in the Bodleian Library in Oxford, the importance of the map in our understanding of globalisation in the early seventeenth century has been recognised. One of the unresolved questions is the origin of the map. This paper addresses the question through material evidence provided by a holistic approach using a suite of complementary analytical techniques. The map was examined in situ and non-invasively by a remote spectral imaging instrument (PRISMS) modified for close range imaging, which was followed by a range of complementary techniques applied to a number of detached fragments, though most of the techniques are non-invasive and can be applied to the map directly in the future. The binding medium was found to be a gum, almost certainly gum Arabic, rather than the animal glue commonly used in Chinese paintings. Some of the pigments and their usage were found to be at odds with the common practice in paintings from China. The detection of gum Arabic, a binding medium used by the Europeans, South and West Asians and the use of a mixture of orpiment and indigo, commonly found in European, South and West Asian paintings gives further evidence on the unusual origins of this map. The likely detection of a basic copper chloride, such as atacamite, in the green areas suggests an influence from the South and West Asian rather than the European tradition. Detailed analysis of the various spectral bands of the spectral image cube along with visual inspection of the large scale colour image showed that the map was not fully planned at the beginning but rather painted in stages, at times by trial and error and that it was unfinished. A new hypothesis for the origin of the Selden map in Aceh Sumatra is proposed based on the new evidences

Probing the near infrared stellar population of Seyfert galaxies

We employ IRTF SpeX NIR (0.8-2.4 microns) spectra to investigate the stellar population (SP), active galactic nuclei (AGN) featureless continuum (FC) and hot dust properties in 9 Sy 1 and 15 Sy 2 galaxies. Both the starlight code and the hot dust as an additional base element were used for the first time in this spectral range. We found evidence of correlation among the equivalent widths (W) Si I 1.59 microns x Mg I 1.58 microns, equally for both kinds of activity. Part of the W{Na I 2.21 microns} and W {CO 2.3 microns} strengths may be related to galaxy inclination. Our synthesis shows significant differences between Sy 1 and Sy 2 galaxies: the hot dust component is required to fit the K-band spectra of ~90% of the Sy 1 galaxies, and only of ~25% of the Sy 2; about 50 % of the Sy 2 galaxies require a $FC$ component contribution >20%, while this fraction increases to 60% in the Sy 1; also, in about 50 % of the Sy2, the combined FC and young components contribute with more than 20%, while this occurs in 90% of the Sy1, suggesting recent star formation in the central region. The central few hundred parsecs of our galaxy sample contain a substantial fraction of intermediate-age SPs with a mean metallicity near solar. Our SP synthesis confirms that the 1.1 micron CN band can be used as a tracer of intermediate-age SPs. The simultaneous fitting of SP, FC and hot dust components increased in ~150% the number of AGNs with hot dust detected and the mass estimated. The NIR emerges as an excellent window to study the stellar population of Sy 1 galaxies, as opposed to the usually heavily attenuated optical range. Our approach opens a new way to investigate and quantify the individual contribution of the three most important NIR continuum components observed in AGNs.Comment: The paper contains 14 figures and 5 tables. Accepted for publication in MNRA

Evidence that MEK1 positively promotes interhomologue double-strand break repair

During meiosis there is an imperative to create sufficient crossovers for homologue segregation. This can be achieved during repair of programmed DNA double-strand breaks (DSBs), which are biased towards using a homologue rather than sister chromatid as a repair template. Various proteins contribute to this bias, one of which is a meiosis specific kinase Mek1. It has been proposed that Mek1 establishes the bias by creating a barrier to sister chromatid repair, as distinct from enforcing strand invasion with the homologue. We looked for evidence that Mek1 positively stimulates strand invasion of the homologue. This was done by analysing repair of DSBs induced by the VMA1-derived endonuclease (VDE) and flanked by directly repeated sequences that can be used for intrachromatid single-strand annealing (SSA). SSA competes with interhomologue strand invasion significantly more successfully when Mek1 function is lost. We suggest the increase in intrachromosomal SSA reflects an opportunistic default repair pathway due to loss of a MEK1 stimulated bias for strand invasion of the homologous chromosome. Making use of an inhibitor sensitive mek1-as1 allele, we found that Mek1 function influences the repair pathway throughout the first4–5 h of meiosis. Perhaps reflecting a particular need to create bias for successful interhomologue events before chromosome pairing is complete

A mutated dph3 gene causes sensitivity of Schizosaccharomyces pombe cells to cytotoxic agents

Dph3 is involved in diphthamide modification of the eukaryotic translation elongation factor eEF2 and in Elongator-mediated modifications of tRNAs, where a 5-methoxycarbonyl-methyl moiety is added to wobble uridines. Lack of such modifications affects protein synthesis due to inaccurate translation of mRNAs at ribosomes. We have discovered that integration of markers at the msh3 locus of Schizosaccharomyces pombe impaired the function of the nearby located dph3 gene. Such integrations rendered cells sensitive to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. We constructed dph3 and msh3 strains with mutated ATG start codons (ATGmut), which allowed investigating drug sensitivity without potential interference by marker insertions. The dph3- ATGmut and a dph3::loxP-ura4-loxM gene disruption strain, but not msh3-ATGmut, turned out to be sensitive to hydroxyurea and methyl methanesulfonate, likewise the strains with cassettes integrated at the msh3 locus. The fungicide sordarin, which inhibits diphthamide modified eEF2 of Saccharomyces cerevisiae, barely affected survival of wild type and msh3Δ S. pombe cells, while the dph3Δ mutant was sensitive. The msh3-ATG mutation, but not dph3Δ or the dph3-ATG mutation caused a defect in mating-type switching, indicating that the ura4 marker at the dph3 locus did not interfere with Msh3 function. We conclude that Dph3 is required for cellular resistance to the fungicide sordarin and to the cytotoxic drugs hydroxyurea and methyl methanesulfonate. This is likely mediated by efficient translation of proteins in response to DNA damage and replication stress

Bio-inspired CO₂ conversion by iron sulfide catalysts under sustainable conditions

The mineral greigite presents similar surface structures to the active sites found in many modern-day enzymes. We show that particles of greigite can reduce CO2 under ambient conditions into chemicals such as methanol, formic, acetic and pyruvic acid. Our results also lend support to the Origin of Life theory on alkaline hydrothermal vents