Artículos (Genética)
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Artículo Role of inorganic phosphate concentrations in in vitro activity of fosfomycin(Elsevier, 2022-02) Ortiz Padilla, Miriam; Portillo Calderón, Inés María; Maldonado, Natalia; Rodríguez Martínez, José Manuel; De Gregorio Iaria, Belén; Merino Bohórquez, Vicente; Rodríguez-Baño, Jesús; Pascual Hernández, Álvaro; Docobo Pérez, Fernando; Universidad de Sevilla. Departamento de Microbiología; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Farmacología; Universidad de Sevilla. Departamento de Medicina; Instituto de Biomedicina de Sevilla (IBIS); Instituto de Salud Carlos III; European Union (UE); Universidad de Sevilla. BIO116: Genética Bacteriana; Universidad de Sevilla. CTS210: Resistencia a AntimicrobianosObjectives: The objective of this study was to evaluate the in vitro activity of fosfomycin under different physiological concentrations of inorganic phosphate (Pi). Methods: The wild-type BW25113 strain, four isogenic mutants (ΔglpT, ΔuhpT, ΔglpT-uhpT, and ΔphoB) and six clinical isolates of Escherichia coli with different fosfomycin susceptibilities were used. EUCAST breakpoints were used. Susceptibility was evaluated by agar dilution using standard Mueller–Hinton agar (Pi concentration of 1 mM similar to human plasma concentration) and supplemented with Pi (13 and 42 mM, minimum and maximum urinary Pi concentrations) and/or glucose-6-phosphate (25 mg/L). Fosfomycin transporter promoter activity was assayed using PglpT::gfpmut2 or PuhpT::gfpmut2 promoter fusions in standard Mueller–Hinton Broth (MHB), supplemented with Pi (13 or 42 mM) ± glucose-6-phosphate. Fosfomycin activity was quantified, estimating fosfomycin EC50 under different Pi concentrations (1, 13 and 42 mM + glucose-6-phosphate) and in time–kill assays using fosfomycin concentrations of 307 (maximum plasma concentration (Cmax)), 1053 and 4415 mg/L (urine Cmax range), using MHB with 28 mM Pi (mean urine Pi concentration) + 25 mg/L glucose-6-phosphate. Results: All the strains showed decreased susceptibility to fosfomycin linked to increased Pi concentrations: 1–4 log2 dilution differences from 1 to 13 mM, and 1–8 log2 dilution differences at 42 mM Pi. Changes in phosphate concentration did not affect the expression of fosfomycin transporters. By increasing Pi concentrations higher fosfomycin EC50 bacterial viability was observed, except against ΔglpT-uhpT. The increase in Pi reduced the bactericidal effect of fosfomycin. Discussion: Pi variations in physiological fluids may reduce fosfomycin activity against E. coli. Elevated Pi concentrations in urine may explain oral fosfomycin failure in non-wild-type but fosfomycin-susceptible E. coli strains.Artículo Activity of Fosfomycin and Amikacin against Fosfomycin-Heteroresistant Escherichia coli Strains in a Hollow-Fiber Infection Model(ASM Journals, 2021-04-19) Portillo Calderón, I.; Ortiz Padilla, Miriam; De Gregorio Iaria, Belén; Merino Bohórquez, Vicente; Blázquez, J.; Rodríguez-Baño, Jesús; Rodríguez Martínez, José Manuel; Pascual Hernández, Álvaro; Docobo Pérez, Fernando; Universidad de Sevilla. Departamento de Microbiología; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Farmacología; Universidad de Sevilla. Departamento de Medicina; Instituto de Biomedicina de Sevilla (IBIS); Universidad de Sevilla. BIO116: Genética Bacteriana; Universidad de Sevilla. CTS210: Resistencia a AntimicrobianosWe evaluated human-like the efficacy of intravenous doses of fosfomycin of 8 g every 8 h (8 g/Q8h) and of amikacin (15 mg/kg/Q24h) in monotherapy and in combination against six fosfomycin-heteroresistant Escherichia coli isolates using a hollow-fiber infection model (HFIM). Six fosfomycin-heteroresistant E. coli isolates (four with strong mutator phenotype) and the control strain E. coli ATCC 25922 were used. Mutant frequencies for rifampin (100 mg/liter), fosfomycin (50 and 200 mg/liter), and amikacin (32 mg/liter) were determined. Fosfomycin and amikacin MICs were assessed by agar dilution (AD), gradient strip assay (GSA), and broth microdilution (BMD). Fosfomycin and amikacin synergies were studied by checkerboard and time-kill assays at different concentrations. The efficacies of fosfomycin (8 g/Q8h) and amikacin (15 mg/kg/Q24h) alone and in combination were assessed using an HFIM. Five isolates were determined to be resistant to fosfomycin by AD and BMD, but all were determined to be susceptible by GSA. All isolates were determined to be susceptible to amikacin. Antibiotic combinations were synergistic in two isolates, and no antagonism was detected. In time-kill assays, all isolates survived under fosfomycin at 64 mg/liter, although at 307 mg/liter only the normomutators and two hypermutators survived. Four isolates survived under 16 mg/liter amikacin, and none survived at 45 mg/liter. No growth was detected under combination conditions. In HFIM, fosfomycin and amikacin monotherapies failed to sterilize bacterial cultures; however, the combination of fosfomycin and amikacin yielded a rapid eradication. There may be a risk of treatment failure of fosfomycin-heteroresistant E. coli isolates using either amikacin or fosfomycin in monotherapy. These results support that the amikacin-fosfomycin combination can rapidly decrease bacterial burden and prevent the emergence of resistant subpopulations against fosfomycin-heteroresistant strains.Artículo VE-1 Regulation of MAPK Signaling Controls Sexual Development in Neurospora Crassa(American Society for Microbiology, 2024) Cea Sánchez, Sara; Martín Villanueva, Sara; Gutiérrez Pozo, Gabriel; Cánovas López, David; Corrochano Peláez, Luis María; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; National Institutes of Health (NIH)Sexual reproduction in fungi allows genetic recombination and increases genetic diversity, allowing adaptation and survival. The velvet complex is a fungal-specific protein assembly that regulates development, pathogenesis, and secondary metabolism in response to environmental cues, such as light. In Neurospora crassa, this complex comprises VE-1, VE-2, and LAE-1. Deletion of ve-1 or ve-2, but not lae-1, leads to increased conidiation (asexual spore formation) and reduced sexual development. Mutants lacking ve-1 and/or ve-2 are female sterile and male fertile, indicating that a VE-1/VE-2 complex regulates the development of female structures. During sexual development, we observed differential regulation of 2,117 genes in dark and 4,364 genes in light between the wild type and the ∆ve-1 strain. The pheromone response and cell wall integrity pathways were downregulated in the ∆ve-1 mutant, especially in light. Additionally, we found reduced levels of both total and phosphorylated MAK-1 and MAK-2 kinases. In vitro experiments demonstrated the binding of VE-1 and VE-2 to the promoters of mak-1 and mak-2, suggesting a direct regulatory role of VE-1/VE-2 in the transcriptional control of MAPK genes to regulate sexual development. Deletion of the photosensor gene white-collar 1 prevented the light-dependent inhibition of sexual development in the ∆ve-1 mutant by increasing transcription of the pheromone response and cell wall integrity pathway genes to the levels in the dark. Our results support the proposal that the regulation of the MAP kinase pathways by the VE-1/ VE-2 complex is a key element in transcriptional regulation that occurs during sexual development. IMPORTANCE Sexual reproduction generates new gene combinations and novel phenotypic traits and facilitates evolution. Induction of sexual development in fungi is often regulated by environmental conditions, such as the presence of light and nutrients. The velvet protein complex coordinates internal cues and environmental signals to regulate development. We have found that VE-1, a component of the velvet complex, regulates transcription during sexual development in the fungus Neurospora crassa. VE-1 regulates the transcription of many genes, including those involved in mitogen-activated protein kinase (MAPK) signaling pathways that are essential in the regulation of sexual development, and regulates the activity of the MAPK pathway. Our findings provide valuable insights into how fungi respond to environmental signals and integrate them into their reproductive processes.Artículo PIWI proteins and piRNAs: key regulators of stem cell biology(Frontiers Media, 2025-02-06) Claro Linares, Fernando; Rojas Ríos, Patricia; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla; Junta de AndalucíaIn this mini review, we discussed the functional roles of PIWI proteins and their associated small RNAs, piRNAs, in regulating gene expression within stem cell biology. Guided by piRNAs, these proteins transcriptionally and posttranscriptionally repress transposons using mechanisms such as the pingpong amplification cycle and phasing to protect germline genomes. Initially identified in Drosophila melanogaster, the piRNA pathway regulate germline stem cell self-renewal and differentiation via cell-autonomous and non-cellautonomous mechanisms. Precisely, in GSCs, PIWI proteins and piRNAs regulate gene expression by modulating chromatin states and directly influencing mRNA translation. For instance, the PIWI protein Aubergine loaded with piRNAs promotes and represses translation of certain mRNAs to balance self-renewal and differentiation. Thus, the piRNA pathway exhibits dual regulatory roles in mRNA stability and translation, highlighting its context-dependent functions. Moreover, PIWI proteins are essential in somatic stem cells to support the regenerative capacity of highly regenerative species, such as planarians. Similarly, in Drosophila intestinal stem cells, the PIWI protein Piwi regulates metabolic pathways and genome integrity, impacting longevity and gut homeostasis. In this case, piRNAs appear absent in the gut, suggesting piRNAindependent regulatory mechanisms. Together, PIWI proteins and piRNAs demonstrate evolutionary conservation in stem cell regulation, integrating TE silencing and gene expression regulation at chromatin and mRNA levels in somatic and germline lineages. Beyond their canonical roles, emerging evidence reveal their broader significance in maintaining stem cell properties and organismal health under physiological and pathological conditions.Artículo Mutational analysis of the inactivating factors, IF7 and IF17 from Synechocystis sp. PCC 6803: Critical role of arginine amino acid residues for glutamine synthetase inactivation(John Wiley & Sons, 2011-11) Saelices, Lorena; Galmozzi, Carla V.; Florencio Bellido, Francisco Javier; Muro Pastor, María Isabel; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Junta de AndalucíaThe Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) activity is controlled by a process that involves protein-protein interaction with two inactivating factors (IF7 and IF17). IF7 is a natively unfolded, 65-residue-long protein, homologous to the carboxy-terminal region of IF17. Both proteins have abundance of positively charged amino acid residues and a high isoelectric point. In this study, we analyse the IF amino acid residues involved in GS inactivation by a mutational approach, both in vitro and in vivo. The results clearly indicate that the GS-IF complex formation must be determined mainly by electrostatic interactions. We have identified three conserved arginine residues of IF7 and IF17 that are essential for the interaction of these proteins with GS. All these residues map in the homologous region of IFs. Furthermore, in vitro analysis of a truncated IF17 protein without the 82-residue-long amino-terminal part, together with the analysis of a Synechocystis strain expressing a chimeric protein, containing this amino-terminal part of IF17 fused to IF7, demonstrates that amino-terminal region of IF17 mostly confers a higher stability to this protein.Artículo The inactivating factor of glutamine synthetase IF17 Is an intrinsically disordered protein, which folds upon binding to its target(American Chemical Society, 2011-10-12) Saelices, Lorena; Galmozzi, Carla V.; Florencio Bellido, Francisco Javier; Muro Pastor, María Isabel; Neira, José L.; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Bioquímica Vegetal y Biología Molecular; Ministerio de Ciencia e Innovación (MICIN). España; Generalitat Valenciana; Junta de AndalucíaIn cyanobacteria, ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase and glutamate synthase (GOGAT). The activity of Synechocystis sp. PCC 6803 glutamine synthetase type I (GS) is controlled by a post-transcriptional process involving protein-protein interactions with two inactivating factors: the 65-residue-long protein (IF7) and the 149-residue-long one (IF17). The sequence of the C terminus of IF17 is similar to IF7; IF7 is an intrinsically disordered protein (IDP). In this work, we study the structural propensities and affinity for GS of IF17 and a chimera protein, IF17N/IF7 (constructed by fusing the first 82 residues of IF17 with the whole IF7) by fluorescence, CD, and NMR. IF17 and IF17N/IF7 are IDPs with residual non-hydrogen-bonded structure, probably formed by α-helical, turn-like, and PPII conformations; several theoretical predictions support these experimental findings. IF17 seems to fold upon binding to GS, as suggested by CD thermal denaturations and steady-state far-UV spectra. The apparent affinity of IF17 for GS, as measured by fluorescence, is slightly smaller (K D ∼1 μM) than that measured for IF7 (∼0.3 μM). The K Ds determined by CD are similar to those measured by fluorescence, but slightly larger, suggesting possible conformational rearrangements in the IFs and/or GS upon binding. Further, the results with IFN17/IF7 suggest that (i) binding of IF17 to the GS is modulated not only by its C-terminal region but also by its N-terminus and (ii) there are weakly structured (that is, "fuzzy") complexes in the ternary GS-IF system.Artículo Evolution of a bistable genetic system in fluctuating and nonfluctuating environments(National Academy of Sciences, 2024-08-30) Fernández Fernández, Rocío; Rodríguez Olivenza, David; Weyer, Esther; Singh, Abhyudai; Casadesús Pursals, Josep; Sánchez Romero, María Antonia; Universidad de Sevilla. Departamento de Microbiología y Parasitología; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). España; Agencia Estatal de Investigación. España; European Union (UE)Epigenetic mechanisms can generate bacterial lineages capable of spontaneously switching between distinct phenotypes. Currently, mathematical models and simulations propose epigenetic switches as a mechanism of adaptation to deal with fluctuating environments. However, bacterial evolution experiments for testing these predictions are lacking. Here, we exploit an epigenetic switch in Salmonella enterica, the opvAB operon, to show clear evidence that OpvAB bistability persists in changing environments but not in stable conditions. Epigenetic control of transcription in the opvAB operon produces OpvABOFF (phage-sensitive) and OpvABON (phage-resistant) cells in a reversible manner and may be interpreted as an example of bet-hedging to preadapt Salmonella populations to the encounter with phages. Our experimental observations and computational simulations illustrate the adaptive value of epigenetic variation as an evolutionary strategy for mutation avoidance in fluctuating environments. In addition, our study provides experimental support to game theory models predicting that phenotypic heterogeneity is advantageous in changing and unpredictable environments.Artículo Genome-wide Function of THO/TREX in Active Genes Prevents R-loop-dependent Replication Obstacles(EMBO Press, 2011) Gómez González, Belén; García Rubio, María Luisa; Bermejo, Rodrigo; Gaillard, Hélène; Shirahige, Katsuhiko; Marín, Antonio; Foiani, Marco; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; Junta de AndalucíaTHO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and prevents transcription-associated recombination. Whether or not it has a ubiquitous role in the genome is unknown. Chromatin immunoprecipitation (ChIP)-chip studies reveal that the Hpr1 component of THO and the Sub2 RNA-dependent ATPase have genome-wide distributions at active ORFs in yeast. In contrast to RNA polymerase II, evenly distributed from promoter to termination regions, THO and Sub2 are absent at promoters and distributed in a gradual 5-2 3-2 gradient. This is accompanied by a genome-wide impact of THO-"Sub2 deletions on expression of highly expressed, long and high G+C-content genes. Importantly, ChIP-chips reveal an over-recruitment of Rrm3 in active genes in THO mutants that is reduced by RNaseH1 overexpression. Our work establishes a genome-wide function for THO-"Sub2 in transcription elongation and mRNP biogenesis that function to prevent the accumulation of transcription-mediated replication obstacles, including R-loops.Artículo XGene expression is circular: Factors for mRNA degradation also foster mRNA synthesis(Elsevier, 2013-05-23) Haimovich, Gal; Medina, Daniel A.; Causse, Sebastien Z.; Garber, Manuel; Millán Zambrano, Gonzalo; Barkai, Oren; Chávez de Diego, Sebastián; Pérez-Ortín, José E.; Darzacq, Xavier; Choder, Mordechai; Universidad de Sevilla. Departamento de Genética; Israel Science Foundation; Ministry of Health, and Rappaport Foundation; Ministerio de Ciencia e Innovación (MICIN). España; Generalitat Valenciana; Junta de Andalucía; European Union (UE); Center for Excellence in Genome ScienceMaintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5′-to-3′ pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin - preferentially ∼30 bp upstream of transcription start-sites - and directly stimulate transcription initiation and elongation. The nuclear role of the decaysome in transcription is linked to its cytoplasmic role in mRNA decay; linkage, in turn, seems to depend on proper shuttling of its components. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected.Artículo Histone post-translational modifications — cause and consequence of genome function(Springer Nature, 2022-03-25) Millán Zambrano, Gonzalo; Burton, Adam; Bannister, Andrew J.; Schneider, Robert; Universidad de Sevilla. Departamento de Genética; German Research Foundation (DFG); Cancer Research. United Kingdom; Wellcome Trust; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE)Much has been learned since the early 1960s about histone post-translational modifications (PTMs) and how they affect DNA-templated processes at the molecular level. This understanding has been bolstered in the past decade by the identification of new types of histone PTM, the advent of new genome-wide mapping approaches and methods to deposit or remove PTMs in a locally and temporally controlled manner. Now, with the availability of vast amounts of data across various biological systems, the functional role of PTMs in important processes (such as transcription, recombination, replication, DNA repair and the modulation of genomic architecture) is slowly emerging. This Review explores the contribution of histone PTMs to the regulation of genome function by discussing when these modifications play a causative (or instructive) role in DNA-templated processes and when they are deposited as a consequence of such processes, to reinforce and record the event. Important advances in the field showing that histone PTMs can exert both direct and indirect effects on genome function are also presented.Artículo A matter of packaging: Influence of nucleosome positioning on heterologous gene expression(Springer Nature, 2011-11-21) Muñoz Centeno, María de la Cruz; Millán Zambrano, Gonzalo; Chávez de Diego, Sebastián; Universidad de Sevilla. Departamento de Genética; Ministerio de Educación y Ciencia (MEC). España; Junta de AndalucíaThe organization of DNA into the various levels of chromatin compaction is the main obstacle that restricts the access of transcriptional machinery to genes. Genome-wide chromatin analyses have shown that there are common chromatin organization patterns for most genes but have also revealed important differences in nucleosome positioning throughout the genome. Such chromatin heterogeneity is one of the reasons why recombinant gene expression is highly dependent on integration sites. Different solutions have been tested for this problem, including artificial targeting of chromatin-modifying factors or the addition of DNA elements, which efficiently counteract the influence of the chromatin environment. An influence of the chromatin configuration of the recombinant gene itself on its transcriptional behavior has also been established. This view is especially important for heterologous genes since the general parameters of chromatin organization change from one species to another. The chromatin organization of bacterial DNA proves particularly dramatic when introduced into eukaryotes. The nucleosome positioning of recombinant genes is the result of the interaction between the machinery of the hosting cell and the sequences of both the recombinant genes and the promoter regions. We discuss the key aspects of this phenomenon from the heterologous gene expression perspective.Artículo R‐loops cause replication impairment and genome instability during meiosis(EMBO Press, 2012) Castellano Pozo, Maikel; García Muse, Tatiana; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia e Innovación (MICIN). España; European Union (UE); Junta de AndalucíaR‐loops are harmful structures with a negative impact on transcription and recombination during mitosis, but no information exists for meiosis. We used Saccharomyces cerevisiae and Caenorhabditis elegans THO mutants as a tool to determine the consequences of R‐loops in meiosis. We found that both S. cerevisiae and C. elegans THO mutants show defective meiosis and an impairment of premeiotic replication as well as DNA‐damage accumulation. Importantly, RNase H partially suppressed the replication impairment and the DNA‐damage accumulation. We conclude that R‐loops can form during meiosis causing replication impairment with deleterious results.Artículo R Loops Are Linked to Histone H3 S10 Phosphorylation and Chromatin Condensation(Cell Press, 2013) Castellano Pozo, Maikel; Santos Pereira, José María; García Rondón, Ana; Barroso Ceballos, Sonia Inés; Andújar, Eloísa; Pérez Alegre, Mónica; García Muse, Tatiana; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Junta de Andalucía; European Union (UE)R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.Artículo The molecular chaperone ALYREF promotes R-loop resolution and maintains genome stability(American Society for Biochemistry and Molecular Biology Inc., 2024-12) Bhandari, Jay; Guillén Mendoza, Cristina; Banks, Kathryn; Eliaz, Lillian; Southwell, Sierra; Eyaa, Darriel; Luna Varo, Rosa María; Aguilera López, Andrés; Xue, Xiaoyu; Universidad de Sevilla. Departamento de Genética; Texas State University; Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC)Unscheduled R-loops usually cause DNA damage and replication stress, and are therefore a major threat to genome stability. Several RNA processing factors, including the conserved THO complex and its associated RNA and DNA-RNA helicase UAP56, prevent R-loop accumulation in cells. Here, we investigate the function of ALYREF, an RNA export adapter associated with UAP56 and the THO complex, in R-loop regulation. We demonstrate that purified ALYREF promotes UAP56-mediated R-loop dissociation in vitro, and this stimulation is dependent on its interaction with UAP56 and R-loops. Importantly, we show that ALYREF binds DNA-RNA hybrids and R-loops. Consistently, ALYREF depletion causes R-loop accumulation and R-loop–mediated genome instability in cells. We propose that ALYREF, apart from its known role in RNA metabolism and export, is a key cellular R-loop coregulator, which binds R-loops and stimulates UAP56-driven resolution of unscheduled R-loops during transcription.Artículo Preparation of a radiobiology beam line at the 18 MeV proton cyclotron facility at CNA(Elsevier, 2020-06) Baratto Roldán, Anna; Jiménez Ramos, María del Carmen; Jimeno González, Sonia; Huertas Sánchez, Pablo; García López, Francisco Javier; Gallardo Fuentes, María Isabel; Cortés Giraldo, Miguel Antonio; Espino Navas, José Manuel; Universidad de Sevilla. Departamento de Economía Aplicada II; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Física Atómica, Molecular y Nuclear; European Union (UE); Ministerio de Ciencia e Innovación (MICIN). España; Junta de AndalucíaProton therapy has gained interest in recent years due to its excellent clinical outcomes. However, the lack of accurate biological data, especially in the Bragg peak region of clinical beams, makes it difficult to implement biophysically optimized treatment plans in clinical practice. In this context, low energy proton accelerator facilities provide the perfect environment to collect good radiobiological data, as they can produce high LET beams with narrow energy distributions. This study presents the radiobiology beam line that has been designed at the 18 MeV proton cyclotron facility at the National Centre of Accelerators (CNA, Seville, Spain), to perform irradiations of mono-layer cell cultures. To ensure that all the cells receive the same dose with a suitable dose rate, low beam intensities and broad and homogeneous beam profiles are necessary. To do so, at the CNA an unfocused beam has been used, broadened with a 500 μm thick aluminium scattering foil. Homogeneous dose profiles, with deviations lower than 10% have been obtained over a circular surface of 35 mm diameter for an incident average energy of 12.8 MeV. Further, a Monte Carlo simulation of the beam line has been developed with Geant4, and benchmarked towards experimental measurements, with differences generally below 1%. Once validated, the code has been used, together with an ionization chamber, for dosimetry studies, to characterize the beam and monitor the dose. Finally, cultures of Human Bone Osteosarcoma cells (U2OS) have been successfully irradiated at the radiobiology beam line, investigating the effects of radiation in terms of DNA damage induction.Artículo Importance of hydrophobic interactions in the single-chained cationic surfactant-DNA complexation(Elsevier, 2018-07-01) López-López, Manuel; López-Cornejo, María del Pilar; Martín, Victoria I.; Ostos Marcos, Francisco José; Checa Rodríguez, Cintia; Prados Carvajal, Rosario; Lebrón Romero, José Antonio; Huertas Sánchez, Pablo; Moyá Morán, María Luisa; Universidad de Sevilla. Departamento de Química Física; Universidad de Sevilla. Departamento de Biología Celular; Universidad de Sevilla. Departamento de Genética; Junta de Andalucía; Universidad de Sevilla; Ministerio de Economía y Competitividad (MINECO). EspañaThe goal of this work was to understand the key factors determining the DNA compacting capacity of single-chained cationic surfactants. Fluorescence, zeta potential, circular dichroism, gel electrophoresis and AFM measurements were carried out in order to study the condensation of the nucleic acid resulting from the formation of the surfactant-DNA complexes. The apparent equilibrium binding constant of the surfactants to the nucleic acid, Kapp, estimated from the experimental results obtained in the ethidium bromide competitive binding experiments, can be considered directly related to the ability of a given surfactant as a DNA compacting agent. The plot of ln(Kapp) vs. ln(cmc), cmc being the critical micelle concentration, for all the bromide and chloride surfactants studied, was found to be a reasonably good linear correlation. This result shows that hydrophobic interactions mainly control the surfactant DNA compaction efficiencyArtículo The bacterial epigenome(Springer nature, 2020-01) Sánchez Romero, María Antonia; Casadesús Pursals, Josep; Universidad de Sevilla. Departamento de Microbiología y Parasitología; Universidad de Sevilla. Departamento de Genética; Ministerio de Ciencia, Innovación y Universidades (MICINN). EspañaIn all domains of life, genomes contain epigenetic information superimposed over the nucleotide sequence. Epigenetic signals control DNA–protein interactions and can cause phenotypic change in the absence of mutation. A nearly universal mechanism of epigenetic signalling is DNA methylation. In bacteria, DNA methylation has roles in genome defence, chromosome replication and segregation, nucleoid organization, cell cycle control, DNA repair and regulation of transcription. In many bacterial species, DNA methylation controls reversible switching (phase variation) of gene expression, a phenomenon that generates phenotypic cell variants. The formation of epigenetic lineages enables the adaptation of bacterial populations to harsh or changing environments and modulates the interaction of pathogens with their eukaryotic hosts.Artículo DNA methylation in bacteria: From the methyl group to the methylome(Elsevier, 2015-06) Sánchez Romero, María Antonia; Cota, Ignacio; Casadesús Pursals, Josep; Universidad de Sevilla. Departamento de Microbiología y Parasitología; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Junta de AndalucíaFormation of C5-methyl-cytosine, N4-methyl-cytosine, and N6-methyl-adenine in bacterial genomes is postreplicative, and occurs at specific targets. Base methylation can modulate the interaction of DNA-binding proteins with their cognate sites, and controls chromosome replication, correction of DNA mismatches, cell cycle-coupled transcription, and formation of epigenetic lineages by phase variation. During four decades, the roles of DNA methylation in bacterial physiology have been investigated by analyzing the contribution of individual methyl groups or small methyl group clusters to the control of DNA-protein interactions. Nowadays, single-molecule real-time sequencing can analyze the DNA methylation of the entire genome (the 'methylome'). Bacterial methylomes provide a wealth of information on the methylation marks present in bacterial genomes, and may open a new era in bacterial epigenomics.Artículo Multiple Roles of the Splicing Complex SF3B in DNA end Resection and Homologous Recombination(Elsevier, 2018) López Saavedra, Ana; Prados Carvajal, Rosario; Cepeda García, Cristina; Jimeno González, Sonia; Huertas Sánchez, Pablo; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). EspañaThe appropriate repair of DNA double strand breaks is critical for genome maintenance. Thus, several cellular pathways collaborate to orchestrate a coordinated response. These include the repair of the breaks, which could be achieved by different mechanisms. A key protein involved in the regulation of the repair of broken chromosomes is CtIP. Here, we have found new partners of CtIP involved in the regulation of DNA break repair through affecting DNA end resection. We focus on the splicing complex SF3B and show that its depletion impairs DNA end resection and hampers homologous recombination. Functionally, SF3B controls CtIP function at, as least, two levels: by affecting CtIP mRNA levels and controlling CtIP recruitment to DNA breaks, in a way that requires ATM-mediated phosphorylation of SF3B2 at serine 289. Indeed, overexpression of CtIP rescues the resection defect caused by SF3B downregulation. Strikingly, other SF3B depletion phenotypes, such as impaired homologous recombination or cellular sensitivity to DNA damaging agents, are independent of CtIP levels, suggesting a more general role of SF3B in controlling the response to chromosome breaks.Artículo The Role of RNA and RNA-related Proteins in the Regulation of DNA Double Strand Break Repair Pathway Choice(Elsevier, 2019) Jimeno González, Sonia; Prados Carvajal, Rosario; Huertas Sánchez, Pablo; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). EspañaDNA end resection is a critical step in the repair of DNA double strand breaks. It controls the way the lesion is going to be repaired, thus its regulation has a great importance in maintaining genomic stability. In this review, we focus in recent discoveries in the field that point to a modulation of resection by RNA molecules and RNA-related proteins. Moreover, we aim to reconcile contradictory reports on the positive or negative effect of DNA:RNA hybrids in the resection process.