Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in

In vivo and in silico determination of essential genes of Campylobacter jejuni

<p>Abstract</p> <p>Background</p> <p>In the United Kingdom, the thermophilic <it>Campylobacter </it>species <it>C. jejuni </it>and <it>C. coli </it>are the most frequent causes of food-borne gastroenteritis in humans. While campylobacteriosis is usually a relatively mild infection, it has a significant public health and economic impact, and possible complications include reactive arthritis and the autoimmune diseases Guillain-Barré syndrome. The rapid developments in "omics" technologies have resulted in the availability of diverse datasets allowing predictions of metabolism and physiology of pathogenic micro-organisms. When combined, these datasets may allow for the identification of potential weaknesses that can be used for development of new antimicrobials to reduce or eliminate <it>C. jejuni </it>and <it>C. coli </it>from the food chain.</p> <p>Results</p> <p>A metabolic model of <it>C. jejuni </it>was constructed using the annotation of the NCTC 11168 genome sequence, a published model of the related bacterium <it>Helicobacter pylori</it>, and extensive literature mining. Using this model, we have used <it>in silico </it>Flux Balance Analysis (FBA) to determine key metabolic routes that are essential for generating energy and biomass, thus creating a list of genes potentially essential for growth under laboratory conditions. To complement this <it>in silico </it>approach, candidate essential genes have been determined using a whole genome transposon mutagenesis method. FBA and transposon mutagenesis (both this study and a published study) predict a similar number of essential genes (around 200). The analysis of the intersection between the three approaches highlights the shikimate pathway where genes are predicted to be essential by one or more method, and tend to be network hubs, based on a previously published <it>Campylobacter </it>protein-protein interaction network, and could therefore be targets for novel antimicrobial therapy.</p> <p>Conclusions</p> <p>We have constructed the first curated metabolic model for the food-borne pathogen <it>Campylobacter jejuni </it>and have presented the resulting metabolic insights. We have shown that the combination of <it>in silico </it>and <it>in vivo </it>approaches could point to non-redundant, indispensable genes associated with the well characterised shikimate pathway, and also genes of unknown function specific to <it>C. jejuni</it>, which are all potential novel <it>Campylobacter </it>intervention targets.</p

Arabidopsis Qc‑SNARE genes BET11 and BET12 are required for fertility and pollen tube elongation

ORCID IDs: 0000-0003-1729-0561 (P.B.-V.); 0000-0003-3459-1331 (G.-Y.J.)Pollen tubes are rapidly growing specialized structures that elongate in a polar manner. They play a crucial role in the delivery of sperm cells through the stylar tissues of the flower and into the embryo sac, where the sperm cells are released to fuse with the egg cell and the central cell to give rise to the embryo and the endosperm. Polar growth at the pollen tube tip is believed to result from secretion of materials by membrane trafficking mechanisms. In this study, we report the functional characterization of Arabidopsis BET11 and BET12, two genes that may code for Qc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). Double mutants (bet11/bet12) in a homozygous/heterozygous background showed reduced transmission of the mutant alleles, reduced fertilization of seeds, defective embryo development, reduced pollen tube lengths and formation of secondary pollen tubes. Both BET11 and BET12 are required for fertility and development of pollen tubes in Arabidopsis. More experiments are required to dissect the mechanisms involved.Academia Sinica (Taiwan)National Science and Technology Program for Agricultural Biotechnology (NSTP/AB, 098S0030055-AA), TaiwanNational Science Council (NSF; 99-2321-B-001-036-MY3), TaiwanUniversidad de Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias Agroalimentarias::Estación Experimental Agrícola Fabio Baudrit Moreno (EEAFBM

Influence of impurity on ice crystal nucleation

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Influence of impurity on ice crystal nucleation

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Influence of Arizona Test Dust (ATD) on the freezing process of high altitude clouds

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Windowless in situ Water Condensation on NaCl nanocubes in Environmental TEM. - VIRTUEL

SSCI-VIDE+ATARI:MEME+FCA:EEH:CLH:LMA:TEPInternational audienceObserving liquids in a Transmission Electron Microscope (TEM) has long been impossible owing to basic thermodynamic limitations due to the need for a high vacuum, typically 10-5 mbar or better, within the column of the instrument, making it impossible to maintain a liquid state at room temperature. With the development of dedicated sealed liquid cells mounted on specific specimen holders, Liquid Cell TEM (LCTEM) has become possible about a decade ago, opening a huge range of possible applications in the field of biology, crystal growth or electrochemistry [1]. In parallel, Environmental TEM (ETEM) was also developed [2]; here a partial pressure can be maintained in the pole-pieces gap where the tip of the sample holder, including the sample itself, is inserted, allowing to perform observations under gas without any sealing membranes as needed with the close-cell technology. Such an ‘open-cell’ approach was also developed in Scanning EM (ESEM, e.g. [3]). With these dedicated ETEM or ESEM configurations, observing liquid such as water layers is possible under a partial pressure of a few mbar if the temperature is cooled down close to the dew point in order to insure a thermodynamic equilibrium between the solid, gas and liquid states: for water, the liquid state can effectively been stabilized in a temperature and pressure range of typically 0 to 11°C and 6 to 15 mbar respectively [4], which are conditions easily accessible in ETEM and ESEM. While LCTEM in a close-cell permits to reach atmospheric pressure, thus allowing to observe water at room temperature, it has the drawback of its advantage: the presence of top and bottom sealing membranes makes it very difficult to perform water condensation from a humid atmosphere and to control water vapor states. Such experiments are possible in ‘open-cell’ ESEM [5] and ETEM [6] and enhance our understanding of the hygroscopic behavior of atmospheric aerosol particles that are known to act as cloud condensation nuclei [7]. Hygroscopic growth, deliquescence and efflorescence of model and real) atmospheric nanoparticles can be directly visualized by these techniques. The present contribution aims at establishing conditions under which aerosols can be adequately observed in a Titan ETEM (FEI/TFS). We use a Gatan liquid-nitrogen (LN2) cryo-holder to cool down the specimen around 0°C. We adjust the temperature by mixing LN2 with a controlled quantity of ethanol. For the purpose of this preliminary investigation, we use NaCl nanoparticles, obtained by vaporizing a salt solution onto a classical holey carbon TEM grid, as a model aerosols. Observations were performed at 300 kV under a humid atmosphere generated by pumping a small sealed water reservoir connected to one of the input lines of the ETEM, the pumping being insured by the molecular turbopumps of its vacuum system. The presence of water (vapor) was controlled by the residual gas analyzer equipping the microscope and by Electron Energy-Loss Spectroscopy (EELS). In a first step, and considering the high voltage at which experiments were performed, a control of the electron flux and dose was realized using different illumination settings in order to define safe imaging conditions avoiding noticeable irradiation damage of the nanocrystals (Fig. 1). Then, both water condensation and evaporation have been performed to follow the evolution of NaCl cubes (Fig. 2). Results will be discussed in terms of relationships between percentage of relative humidity and water uptake of the NaCl particles as a function of T and P [8]. References:[1] FM Ross (Ed.), Liquid Cell Electron Microscopy (Advances in Microscopy and Microanalysis), Cambridge University Press, Cambridge (2017), 524 p.[2] TW Hansen, J.B. Wagner (Ed.), Controlled Atmosphere TEM, Springer, New York, (2016), 332 p.[3] A Bogner et al. Micron, 38 (2007) 390. [4] DJ Stokes. Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-ESEM), (2008), John Wiley & Sons Ltd., 221 p.[5] RC Hoffman et al. J. Aerosol Science, 35 7 (2004) 869. [6] ME Wise et al. Aerosol Science & Technology, 39:9 (2005) 849; C Cassidy et al. Plos One, 12 11 (2017) e0186899; BDA Levin et al. Microscopy and Microanal. (2020) 1.[7] U Lohmann et al. An Introduction to Clouds: From the Microscale to Climate, Cambridge University Press, Cambridge, UK (2016), 391 p.[8] The authors acknowledge the Consortium Lyon – St-Etienne de Microscopie (CLYM, www.clym.fr), the Centre Technologique des Microstructures (http://microscopies.univ-lyon1.fr/) for practical assistance and the French National Research Agency (ANR, www.anr.fr) for supporting project ANR-20-CE42-0008

Windowless in situ Water Condensation on NaCl nanocubes in Environmental TEM. - VIRTUEL

SSCI-VIDE+ATARI:MEME+FCA:EEH:CLH:LMA:TEPInternational audienceObserving liquids in a Transmission Electron Microscope (TEM) has long been impossible owing to basic thermodynamic limitations due to the need for a high vacuum, typically 10-5 mbar or better, within the column of the instrument, making it impossible to maintain a liquid state at room temperature. With the development of dedicated sealed liquid cells mounted on specific specimen holders, Liquid Cell TEM (LCTEM) has become possible about a decade ago, opening a huge range of possible applications in the field of biology, crystal growth or electrochemistry [1]. In parallel, Environmental TEM (ETEM) was also developed [2]; here a partial pressure can be maintained in the pole-pieces gap where the tip of the sample holder, including the sample itself, is inserted, allowing to perform observations under gas without any sealing membranes as needed with the close-cell technology. Such an ‘open-cell’ approach was also developed in Scanning EM (ESEM, e.g. [3]). With these dedicated ETEM or ESEM configurations, observing liquid such as water layers is possible under a partial pressure of a few mbar if the temperature is cooled down close to the dew point in order to insure a thermodynamic equilibrium between the solid, gas and liquid states: for water, the liquid state can effectively been stabilized in a temperature and pressure range of typically 0 to 11°C and 6 to 15 mbar respectively [4], which are conditions easily accessible in ETEM and ESEM. While LCTEM in a close-cell permits to reach atmospheric pressure, thus allowing to observe water at room temperature, it has the drawback of its advantage: the presence of top and bottom sealing membranes makes it very difficult to perform water condensation from a humid atmosphere and to control water vapor states. Such experiments are possible in ‘open-cell’ ESEM [5] and ETEM [6] and enhance our understanding of the hygroscopic behavior of atmospheric aerosol particles that are known to act as cloud condensation nuclei [7]. Hygroscopic growth, deliquescence and efflorescence of model and real) atmospheric nanoparticles can be directly visualized by these techniques. The present contribution aims at establishing conditions under which aerosols can be adequately observed in a Titan ETEM (FEI/TFS). We use a Gatan liquid-nitrogen (LN2) cryo-holder to cool down the specimen around 0°C. We adjust the temperature by mixing LN2 with a controlled quantity of ethanol. For the purpose of this preliminary investigation, we use NaCl nanoparticles, obtained by vaporizing a salt solution onto a classical holey carbon TEM grid, as a model aerosols. Observations were performed at 300 kV under a humid atmosphere generated by pumping a small sealed water reservoir connected to one of the input lines of the ETEM, the pumping being insured by the molecular turbopumps of its vacuum system. The presence of water (vapor) was controlled by the residual gas analyzer equipping the microscope and by Electron Energy-Loss Spectroscopy (EELS). In a first step, and considering the high voltage at which experiments were performed, a control of the electron flux and dose was realized using different illumination settings in order to define safe imaging conditions avoiding noticeable irradiation damage of the nanocrystals (Fig. 1). Then, both water condensation and evaporation have been performed to follow the evolution of NaCl cubes (Fig. 2). Results will be discussed in terms of relationships between percentage of relative humidity and water uptake of the NaCl particles as a function of T and P [8]. References:[1] FM Ross (Ed.), Liquid Cell Electron Microscopy (Advances in Microscopy and Microanalysis), Cambridge University Press, Cambridge (2017), 524 p.[2] TW Hansen, J.B. Wagner (Ed.), Controlled Atmosphere TEM, Springer, New York, (2016), 332 p.[3] A Bogner et al. Micron, 38 (2007) 390. [4] DJ Stokes. Principles and Practice of Variable Pressure/Environmental Scanning Electron Microscopy (VP-ESEM), (2008), John Wiley & Sons Ltd., 221 p.[5] RC Hoffman et al. J. Aerosol Science, 35 7 (2004) 869. [6] ME Wise et al. Aerosol Science & Technology, 39:9 (2005) 849; C Cassidy et al. Plos One, 12 11 (2017) e0186899; BDA Levin et al. Microscopy and Microanal. (2020) 1.[7] U Lohmann et al. An Introduction to Clouds: From the Microscale to Climate, Cambridge University Press, Cambridge, UK (2016), 391 p.[8] The authors acknowledge the Consortium Lyon – St-Etienne de Microscopie (CLYM, www.clym.fr), the Centre Technologique des Microstructures (http://microscopies.univ-lyon1.fr/) for practical assistance and the French National Research Agency (ANR, www.anr.fr) for supporting project ANR-20-CE42-0008

Virulence et effets des antibiotiques chez les staphylocoques à coagulase négatives responsables de bacteriémies liées au cathéter

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