Artículos (Genética)

URI permanente para esta colecciónhttps://hdl.handle.net/11441/10879

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A Carotenogenic Enzyme Aggregate in Phycomyces: Evidence from Quantitive Complementation
(National Academy of Sciences, 1971) Guardia, M.D. de la; González Aragón, Carlos; Murillo, F. Javier; Cerdá Olmedo, Enrique; Universidad de Sevilla. Departamento de Genética
Wild-type Phycomyces blakesleeanus accumulates β-carotene, while the mutant strain C2 is unable to synthesize carotenoids, and the mutant strain C9 accumulates lycopene. Heterokaryons containing a proportion, p, of C2 nuclei and (l — p) of C9 nuclei accumulate lycopene, γ-carotene, and β-carotene in the relative amounts (l — p), p(l — p), and p2, respectively, for different values of p. It is shown that these results are expected from the operation of a carotenogenic enzyme aggregate that works as an assembly line and contains two copies of a cyclase, which is defective in strain C9, as well as other enzymes.
A complete set of nascent transcription rates for yeast genes
(2010) Chávez de Diego, Sebastián; Pérez Ortín, José E.; Pelechano, Vicent
A eukaryotic-like 3â€² untranslated region in Salmonella enterica hilD mRNA
(Oxford University Press, 2014) López Garrido, Javier; Puerta Fernández, Elena; Casadesús Pursals, Josep; Universidad de Sevilla. Departamento de Genética; Ministerio de Economía y Competitividad (MINECO). España; Junta de Andalucía
Long 3' untranslated regions (3'UTRs) are common in eukaryotic mRNAs. In contrast, long 3'UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3'UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3'UTR increases the hilD mRNA level, suggesting that the hilD 3'UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3'UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3'UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3'UTR mRNAs, suggesting that the hilD 3'UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3'UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3'UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3'UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.
A functional connection between translation elongation and protein folding at the ribosome exit tunnel in Saccharomyces cerevisiae
(Oxford University Press, 2021) Rodríguez Galán, Olga; García Gómez, Juan José; Valle Rosado, Iván; Wei, Wu; Méndez-Godoy, Alfonso; Pillet, Benjamin; Alekseenko, Alisa; Steinmetz, Lars M.; Pelechano, Vicent; Kressler, Dieter; Cruz Díaz, Jesús de la; Universidad de Sevilla. Departamento de Genética
Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5′ region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.
A gene-specific requirement for FACT during transcription is related to the chromatin organization of the transcribed region
(2006) Jimeno González, Silvia; Gómez Herreros, Fernando; Alepuz, Paula M.; Chávez de Diego, Sebastián; Universidad de Sevilla. Departamento de Genética; Ministerio de Educación y Ciencia (MEC). España; Junta de Andalucía
The FACT complex stimulates transcription elongation on nucleosomal templates. In vivo experiments also involve FACT in the reassembly of nucleosomes traversed by RNA polymerase II. Since several features of chromatin organization vary throughout the genome, we wondered whether FACT is equally required for all genes. We show in this study that the in vivo depletion of Spt16, one of the subunits of Saccharomyces cerevisiae FACT, strongly affects transcription of three genes, GAL1, PHO5, and Kluyveromyces lactis LAC4, which exhibit positioned nucleosomes at their transcribed regions. In contrast, showing a random nucleosome structure, YAT1 and Escherichia coli lacZ are only mildly influenced by Spt16 depletion. We also show that the effect of Spt16 depletion on GAL1 expression is suppressed by a histone mutation and that the insertion of a GAL1 fragment, which allows the positioning of two nucleosomes, at the 5′ end of YAT1 makes the resulting transcription unit sensitive to Spt16 depletion. These results indicate that FACT requirement for transcription depends on the chromatin organization of the 5′ end of the transcribed region.
A genome-wide function of THSC/TREX-2 at active genes prevents transcription–replication collisions
(Oxford University Press, 2014) Santos Pereira, José María; García Rubio, María Luisa; González Aguilera, Cristina; Luna Varo, Rosa María; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética
The THSC/TREX-2 complex of Saccharomyces cerevisiae mediates the anchoring of transcribed genes to the nuclear pore, linking transcription elongation with mRNA export and genome stability, as shown for specific reporters. However, it is still unknown whether the function of TREX-2 is global and the reason for its relevant role in genome integrity. Here, by studying two TREX-2 representative subunits, Thp1 and Sac3, we show that TREX-2 has a genome-wide role in gene expression. Both proteins show similar distributions along the genome, with a gradient disposition at active genes that increases towards the 3′ end. Thp1 and Sac3 have a relevant impact on the expression of long, G+C-rich and highly transcribed genes. Interestingly, replication impairment detected by the genome-wide accumulation of the replicative Rrm3 helicase is increased preferentially at highly expressed genes in the thp1Δ and sac3Δ mutants analyzed. Therefore, our work provides evidence of a function of TREX-2 at the genome-wide level and suggests a role for TREX-2 in preventing transcription–replication conflicts, as a source of genome instability derived from a defective messenger ribonucleoprotein particle (mRNP) biogenesis.
A Genome-Wide Screen Identifies Yeast Genes Required for Tolerance to Technical Toxaphene, an Organochlorinated Pesticide Mixture
(2013) Gaytán, B. D.; Lerot, J. M.; Chávez de Diego, Sebastián; Peñate Salas, Xenia; Loguinov, A.; Denslow, N.; Vulpe, C.; Universidad de Sevilla. Departamento de Genética
A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection
(Nature Publishing Group, 2016) López Saavedra, Ana; Gómez Cabello, Daniel; Domínguez Sánchez, María Salud; Mejías Navarro, Fernando; Fernández Ávila, María Jesús; Martínez Macías, María Isabel; Huertas Sánchez, Pablo; Universidad de Sevilla. Departamento de Genética
There are two major and alternative pathways to repair DNA double-strand breaks: non-homologous end-joining and homologous recombination. Here we identify and characterize novel factors involved in choosing between these pathways; in this study we took advantage of the SeeSaw Reporter, in which the repair of double-strand breaks by homology-independent or -dependent mechanisms is distinguished by the accumulation of green or red fluorescence, respectively. Using a genome-wide human esiRNA (endoribonuclease-prepared siRNA) library, we isolate genes that control the recombination/end-joining ratio. Here we report that two distinct sets of genes are involved in the control of the balance between NHEJ and HR: those that are required to facilitate recombination and those that favour NHEJ. This last category includes CCAR2/DBC1, which we show inhibits recombination by limiting the initiation and the extent of DNA end resection, thereby acting as an antagonist of CtIP.
A glimpse into the basis of vision in the kingdom Mycota
(Elsevier, 2010) Idnurm, Alexander; Verma, Surbhi; Corrochano Peláez, Luis María; Universidad de Sevilla. Departamento de Genética; National Institutes of Health. United States; National Science Foundation (NSF). United States; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía
Virtually all organisms exposed to light are capable of sensing this environmental signal. In recent years the photoreceptors that mediate the ability of fungi to " see" have been identified in diverse species, and increasingly characterized. The small sizes of fungal genomes and ease in genetic and molecular biology manipulations make this kingdom ideal amongst the eukaryotes for understanding photosensing. The most widespread and conserved photosensory protein in the fungi is White collar 1 (WC-1), a flavin-binding photoreceptor that functions with WC-2 as a transcription factor complex. Other photosensory proteins in fungi include opsins, phytochromes and cryptochromes whose roles in fungal photobiology are not fully resolved and their distribution in the fungi requires further taxon sampling. Additional unknown photoreceptors await discovery. This review discusses the effects of light on fungi and the evolutionary processes that may have shaped the ability of species to sense and respond to this signal.
A global perspective on carotenoids: metabolism, biotechnology, and benefits for nutrition and health.
(Elsevier, 2018-04-04) Rodríguez Concepción, Manuel; Ávalos Cordero, Francisco Javier; Bonet Piña, María Luisa; Boronat Margosa, Albert; Gómez Gómez, Lourdes; Hornero Méndez, Dámaso; Limón Mirón, María del Carmen; Meléndez Martínez, Antonio Jesús; Olmedilla Alonso, Begoña; Universidad de Sevilla. Departamento de Genética; Universidad de Sevilla. Departamento de Nutrición y Bromatología, Toxicología y Medicina Legal
Carotenoids are lipophilic isoprenoid compounds synthesized by all photosynthetic organisms and some non-photosynthetic bacteria and fungi. With some notable exceptions, animals (including humans) do not produce carotenoids de novo but take them in their diets. In photosynthetic systems carotenoids are essential for photoprotection against excess light and contribute to light harvesting, but perhaps they are best known for their properties as natural pigments in the yellow to red range. Carotenoids can be associated to fatty acids, sugars, proteins, or other compounds that can change their physical and chemical properties and influence their biological roles. Furthermore, oxidative cleavage of carotenoids produces smaller molecules such as apocarotenoids, some of which are important pigments and volatile (aroma) compounds. Enzymatic breakage of carotenoids can also produce biologically active molecules in both plants (hormones, retrograde signals) and animals (retinoids). Both carotenoids and their enzymatic cleavage products are associated with other processes positively impacting human health. Carotenoids are widely used in the industry as food ingredients, feed additives, and supplements. This review, contributed by scientists of complementary disciplines related to carotenoid research, covers recent advances and provides a perspective on future directions on the subjects of carotenoid metabolism, biotechnology, and nutritional and health benefits.
A Live Salmonella Vaccine Delivering PcrV through the Type III Secretion System Protects against Pseudomonas aeruginosa
(American Society for Microbiology, 2019) Aguilera Herce, Julia; García Quintanilla, Meritxell de Jesús; McConnell, MJ; Ramos Morales, Francisco; Romero Flores, Rocío; Universidad de Sevilla. Departamento de Genética
Pseudomonas aeruginosa is a common Gram-negative opportunistic pathogen that is intrinsically resistant to a wide range of antibiotics. The development of a broadly protective vaccine against P. aeruginosa remains a major challenge. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express P. aeruginosa antigens. A fusion between the S. enterica type III secretion effector protein SseJ and the P. aeruginosa antigen PcrV expressed under the control of the sseA promoter was translocated by Salmonella into host cells in vitro and elicited the generation of specific antibodies in mice. Mice immunized with attenuated Salmonella expressing this fusion had reduced bacterial loads in the spleens and lungs and lower serum levels of proinflammatory cytokines than control mice after P. aeruginosa infection. Importantly, immunized mice also showed significantly enhanced survival in this model. These results suggest that type III secretion effectors of S. enterica are appropriate carriers in the design of a live vaccine to prevent infections caused by P. aeruginosa IMPORTANCE The Gram-negative bacterium Pseudomonas aeruginosa is an important opportunistic pathogen that causes infections in cystic fibrosis and hospitalized patients. Therapeutic treatments are limited due to the emergence and spread of new antibiotic-resistant strains. In this context, the development of a vaccine is a priority. Here, we used an attenuated strain of Salmonella enterica serovar Typhimurium as a vehicle to express and deliver the Pseudomonas antigen PcrV. This vaccine induced the generation of specific antibodies in mice and protected them from lethal infections with P. aeruginosa This is an important step toward the development of an effective vaccine for the prevention of infections caused by P. aeruginosa in humans
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 Andalucía
The 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.
A new challenge for data analytics: transposons
(BMC, 2022) Wellinger, Ralf Erik; Aguilar Ruiz, Jesús Salvador; Universidad de Sevilla. Departamento de Genética
A new connection of mRNP biogenesis and export with transcription-coupled repair
(Oxford University Press, 2007) Gaillard, Hélène; Wellinger, Ralf Erik; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética; Ministerio de Educación y Ciencia (MEC). España; Junta de Andalucía
Although DNA repair is faster in the transcribed strand of active genes, little is known about the possible contribution of mRNP biogenesis and export in transcription-coupled repair (TCR). Interestingly, mutants of THO, a transcription complex involved in maintenance of genome integrity, mRNP biogenesis and export, were recently found to be deficient in nucleotide excision repair. In this study we show by molecular DNA repair analysis, that Sub2-Yra1 and Thp1-Sac3, two main mRNA export complexes, are required for efficient TCR in yeast. Careful analysis revealed that THO mutants are also specifically affected in TCR. Ribozyme-mediated mRNA self-cleavage between two hot spots for UV damage showed that efficient TCR does not depend on the nascent mRNA, neither in wild-type nor in mutant cells. Along with severe UV damage-dependent loss in processivity, RNAPII was found binding to chromatin upon UV irradiation in THO mutants, suggesting that RNAPII remains stalled at DNA lesions. Furthermore, Def1, a factor responsible for the degradation of stalled RNAPII, appears essential for the viability of THO mutants subjected to DNA damage. Our results indicate that RNAPII is not proficient for TCR in mRNP biogenesis and export mutants, opening new perspectives on our knowledge of TCR in eukaryotic cells.
A new interaction between BRCA2 and DDX5 promotes the repair of DNA breaks at transcribed chromatin
(Taylor & Francis, 2021) Gómez González, Belén; Sessa, Gaetana; Carreira, Aura; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética
In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients.
A new role for Rrm3 in repair of replication-born DNA breakage by sister chromatid recombination
(Public Library of Science, 2017) Muñoz Galván, Sandra; García Rubio, María Luisa; Ortega Moreno, Pedro; Ruiz Pérez, José Francisco; Jimeno González, Sonia; Pardo, Benjamín; Gómez González, Belén; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética
Replication forks stall at different DNA obstacles such as those originated by transcription. Fork stalling can lead to DNA double-strand breaks (DSBs) that will be preferentially repaired by homologous recombination when the sister chromatid is available. The Rrm3 helicase is a replisome component that promotes replication upon fork stalling, accumulates at highly transcribed regions and prevents not only transcription-induced replication fork stalling but also transcription-associated hyper-recombination. This led us to explore the possible role of Rrm3 in the repair of DSBs when originating at the passage of the replication fork. Using a mini-HO system that induces mainly single-stranded DNA breaks, we show that rrm3Δ cells are defective in DSB repair. The defect is clearly seen in sister chromatid recombination, the major repair pathway of replication-born DSBs. Our results indicate that Rrm3 recruitment to replication-born DSBs is crucial for viability, uncovering a new role for Rrm3 in the repair of broken replication forks.
A novel approach for organelle-specific DNA damage targeting reveals different susceptibility of mitochondrial DNA to the anticancer drugs camptothecin and topotecan
(2009) Wellinger, Ralf Erik; Díaz de la Loza, María del Carmen; Universidad de Sevilla. Departamento de Genética
A novel class of Mrna-containing cytoplasmic granules are produced in response to UV-irradiation
(American Society for Cell Biology, 2008) Gaillard, Hélène; Aguilera López, Andrés; Universidad de Sevilla. Departamento de Genética
Nucleic acids are substrates for different types of damage, but little is known about the fate of damaged RNAs. We addressed the existence of an RNA-damage response in yeast. The decay kinetics of GAL1p-driven mRNAs revealed a dose-dependent mRNA stabilization upon UV-irradiation that was not observed after heat or saline shocks, or during nitrogen starvation. UV-induced mRNA stabilization did not depend on DNA repair, damage checkpoint or mRNA degradation machineries. Notably, fluorescent in situ hybridization revealed that after UV-irradiation, polyadenylated mRNA accumulated in cytoplasmic foci that increased in size with time. In situ colocalization showed that these foci are not processing-bodies, eIF4E-, eIF4G-, and Pab1-containing bodies, stress granules, autophagy vesicles, or part of the secretory or endocytic pathways. These results point to the existence of a specific eukaryotic RNA-damage response, which leads to new polyadenylated mRNA-containing granules (UV-induced mRNA granules; UVGs). We propose that potentially damaged mRNAs, which may be deleterious to the cell, are temporarily stored in UVG granules to safeguard cell viability.
A novel endo-β-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum
(American Society for Microbiology, 1995) Cruz Díaz, Jesús de la; Pintor Toro, José Antonio; Benítez Fernández, Concepción Tahía; Llobell González, Antonio; Romero González, Luis Carlos; Universidad de Sevilla. Departamento de Genética
The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular β-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for β-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this β-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls
A novel lncRNA as a positive regulator of carotenoid biosynthesis in Fusarium
(Nature Research, 2020) Parra Rivero, Obdulia; Pardo Medina, Javier; Gutiérrez Pozo, Gabriel; Limón Mirón, María del Carmen; Ávalos Cordero, Francisco Javier; Universidad de Sevilla. Departamento de Genética
The fungi Fusarium oxysporum and Fusarium fujikuroi produce carotenoids, lipophilic terpenoid pigments of biotechnological interest, with xanthophyll neurosporaxanthin as the main end product. Their carotenoid biosynthesis is activated by light and negatively regulated by the RING-finger protein CarS. Global transcriptomic analysis identified in both species a putative 1-kb lncRNA that we call carP, referred to as Fo-carP and Ff-carP in each species, upstream to the gene carS and transcribed from the same DNA strand. Fo-carP and Ff-carP are poorly transcribed, but their RNA levels increase in carS mutants. The deletion of Fo-carP or Ff-carP in the respective species results in albino phenotypes, with strong reductions in mRNA levels of structural genes for carotenoid biosynthesis and higher mRNA content of the carS gene, which could explain the low accumulation of carotenoids. Upon alignment, Fo-carP and Ff-carP show 75-80% identity, with short insertions or deletions resulting in a lack of coincident ORFs. Moreover, none of the ORFs found in their sequences have indications of possible coding functions. We conclude that Fo-carP and Ff-carP are regulatory lncRNAs necessary for the active expression of the carotenoid genes in Fusarium through an unknown molecular mechanism, probably related to the control of carS function or expression

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