scholarly journals Análisis de componentes de gránulos ribonucleoproteicos en Drosophila

2012 ◽  
Author(s):  
◽  
Carla Layana
Keyword(s):  
Drosophila Melanogaster ◽  
Stress Granules ◽  
Processing Bodies ◽  
Expresión Génica

La regulación de la expresión génica es un proceso altamente regulado, que controla qué genes son expresados en cada momento. Uno de los controles más dinámicos del proteoma celular es la regulación de la traducción. El control de la expresión de los mensajeros celulares se ha relacionado hace algunos años con dos estructuras de silenciamiento particulares, los PB (de processing bodies) y los SG (de stress granules). Ambas son formaciones citoplasmáticas que acumulan ARNm y proteínas. Los PB son estructuras constitutivas de las células mientras que los SG aparecen frente a estímulos de estrés celular. En la primera parte de esta tesis se realizó una caracterización de los PB en células S2 Drosophila melanogaster. Particularmente demostramos la presencia de las proteínas Lsm-1, Me31B y eIF4E en los PB. Durante estrés inducido por arsenito de sodio se encontró a eIF4E en SG. Se realizó un estudio más minucioso sobre eIF4E. El análisis de diversos mutantes demostró que el residuo W117, parte del dominio de unión a las 4E-BP, es el responsable de la ubicación de este factor en PB y SG. Mientras que los residuos que participan en la unión al cap de los mensajeros (W100 y W 146) no están involucrados en este proceso. Finalmente se realizó el estudio de interacciones entre estos componentes en PB de células S2 in vivo mediante FRET. Se demostró que las proteínas Lsm-1 y Me31B interaccionan con eIF4E. Sin embargo no pudo ser demostrada por este método la interacción entre Me31B y Lsm-1. Se realizó la predicción de posibles sitios de interacción entre eIF4E y Me31B mediante modelado por homología.

scholarly journals Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

2005 ◽  
Vol 169 (6) ◽  
pp. 871-884 ◽  
Author(s):  
Nancy Kedersha ◽  
Georg Stoecklin ◽  
Maranatha Ayodele ◽  
Patrick Yacono ◽  
Jens Lykke-Andersen ◽  
...  
Keyword(s):  
Mrna Decay ◽  
Stress Granules ◽  
Mrna Degradation ◽  
Processing Bodies ◽  
Eif2α Phosphorylation ◽  
Protein Components

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2α phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

scholarly journals Dinámica citoplasmática de mRNAs como mecanismo de regulación post-transcripcional de la expresión génica durante la simbiosis fijadora de nitrógeno

2019 ◽  
Author(s):  
◽  
Marcos Lancia
Keyword(s):  
Stress Granules ◽  
Nod Factor ◽  
P Bodies ◽  
Expresión Génica

La disponibilidad de nitrógeno es uno de los factores más importantes que limita el crecimiento y desarrollo de las plantas. La interacción simbiótica entre leguminosas y bacterias del suelo fijadoras de nitrógeno pertenecientes a la familia Rhizobiaceae resulta en la formación de un nuevo órgano, denominado nódulo, donde la bacteria fija el nitrógeno atmosférico (FBN) a formas reducidas que pueden ser incorporadas al metabolismo de la planta. El establecimiento de la simbiosis requiere una reprogramación de la expresión génica de las células de la raíz que involucra cambios a nivel transcripcional y postranscripcional. Dentro de estos últimos se encuentra la distribución dinámica de los mRNAs en distintos complejos ribonucleoproteicos (RNPs) citoplasmáticos, principalmente los polisomas, los cuerpos de procesamiento (P-bodies o PBs) y los gránulos de estrés (Stress Granules o SGs). En esta tesis doctoral se desarrolló una técnica de purificación de complejos RNPs en la planta leguminosa M. truncatula, que permita caracterizar el movimiento de RNAs entre los diferentes complejos RNP citoplasmáticos durante la simbiosis. La expresión de las proteínas DCP1 y RBP47 fusionadas al tag FLAG permitió co-inmunopurificar el RNA asociado a PBs y SGs, respectivamente. La cuantificación de mRNAs de genes involucrados en la vía de señalización del Nod Factor (NF) mostró que existe una regulación postranscripcional de los genes de la vía del NF en M. truncatula que depende de la movilización dinámica de RNAs entre los polisomas, los SGs y los PBs. Por otra parte, la expresión de las fusiones RBP47-RFP y DCP1-YFP permitieron visualizar la dinámica y colocalización de los RNPs in vivo en tejido foliar de N. benthamiana y de raíces de M. truncatula. En el segundo capítulo, se analizaron las características de los mRNAs que presentaban un cambio en la eficiencia traduccional en respuesta a la inoculación con rizobios. La longitud de los mRNAs, el contenido GC, y la estructura secundaria, tanto de sus secuencias no traducidas como de las codificantes, están correlacionadas con la eficiencia traduccional, sugiriendo que estos parámetros influyen en la asociación de los transcriptos a los polisomas en el contexto de la reprogramación génica y la modulación de su traducción durante la simbiosis. En el tercer capítulo, se muestra que las redes transcripcionales que operan en la simbiosis son diferentes a las que participan en la selección de aquellas cepas más eficientes en la simbiosis. Estas diferencias involucran elementos de regulación que actúan tanto a nivel transcripcional como postranscripcional. Los resultados obtenidos en este trabajo de tesis han arrojado luz sobre la dinámica citoplasmática de los mRNAs y han permitido caracterizar los elementos regulatorios del DNA y del RNA asociados a la simbiosis fijadora de nitrógeno, sentando las bases para futuros proyectos de investigación y desarrollo que permitan optimizar la fijación de nitrógeno en leguminosas de interés agronómico.

Insights into amyloid disease from fly models

2014 ◽  
Vol 56 ◽  
pp. 69-83 ◽  
Author(s):  
Ko-Fan Chen ◽  
Damian C. Crowther
Keyword(s):  
Drosophila Melanogaster ◽  
Neurodegenerative Disorders ◽  
Amyloid Β ◽  
Amyloid Β Peptide ◽  
Disease Pathogenesis ◽  
Amyloid Aggregates ◽  
Aβ Amyloid ◽  
Polypeptide Chains ◽  
Amyloid Diseases

The formation of amyloid aggregates is a feature of most, if not all, polypeptide chains. In vivo modelling of this process has been undertaken in the fruitfly Drosophila melanogaster with remarkable success. Models of both neurological and systemic amyloid diseases have been generated and have informed our understanding of disease pathogenesis in two main ways. First, the toxic amyloid species have been at least partially characterized, for example in the case of the Aβ (amyloid β-peptide) associated with Alzheimer's disease. Secondly, the genetic underpinning of model disease-linked phenotypes has been characterized for a number of neurodegenerative disorders. The current challenge is to integrate our understanding of disease-linked processes in the fly with our growing knowledge of human disease, for the benefit of patients.

scholarly journals Synthesis, Quantification, and Characterization of Fatty Acid Amides from In Vitro and In Vivo Sources

Molecules ◽  
2021 ◽  
Vol 26 (9) ◽  
pp. 2543
Author(s):  
Ruidong Ni ◽  
Suzeeta Bhandari ◽  
Perry R. Mitchell ◽  
Gabriela Suarez ◽  
Neel B. Patel ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Fatty Acid ◽  
Apis Mellifera ◽  
Chemical Synthesis ◽  
Acid Amide ◽  
Fatty Acid Amides ◽  
Fatty Acid Amide

Fatty acid amides are a diverse family of underappreciated, biologically occurring lipids. Herein, the methods for the chemical synthesis and subsequent characterization of specific members of the fatty acid amide family are described. The synthetically prepared fatty acid amides and those obtained commercially are used as standards for the characterization and quantification of the fatty acid amides produced by biological systems, a fatty acid amidome. The fatty acid amidomes from mouse N18TG2 cells, sheep choroid plexus cells, Drosophila melanogaster, Bombyx mori, Apis mellifera, and Tribolium castaneum are presented.

scholarly journals In Vivo and In Vitro Assays Evaluating the Biological Activity of Taurine, Glucose and Energetic Beverages

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2198
Author(s):  
Marcos Mateo-Fernández ◽  
Fernando Valenzuela-Gómez ◽  
Rafael Font ◽  
Mercedes Del Río-Celestino ◽  
Tania Merinas-Amo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drosophila Melanogaster ◽  
Biological Activity ◽  
Biological Effects ◽  
Methylation Status ◽  
Repetitive Sequences ◽  
Energy Drinks ◽  
Red Bull

Taurine is one of the main ingredients used in energy drinks which are highly consumed in adolescents for their sugary taste and stimulating effect. With energy drinks becoming a worldwide phenomenon, the biological effects of these beverages must be evaluated in order to fully comprehend the potential impact of these products on the health due to the fact nutrition is closely related to science since the population consumes food to prevent certain diseases. Therefore, the aim of this study was to evaluate the biological effects of taurine, glucose, classic Red Bull® and sugar-free Red Bull® in order to check the food safety and the nutraceutical potential of these compounds, characterising different endpoints: (i) Toxicology, antitoxicology, genotoxicology and life expectancy assays were performed in the Drosophila melanogaster model organism; (ii) The in vitro chemopreventive activity of testing compounds was determined by assessing their cytotoxicity, the proapoptotic DNA-damage capability to induce internucleosomal fragmentation, the strand breaks activity and the modulator role on the methylation status of genomic repetitive sequences of HL-60 promyelocytic cells. Whereas none tested compounds showed toxic or genotoxic effect, all tested compounds exerted antitoxic and antigenotoxic activity in Drosophila. Glucose, classic Red Bull® and sugar-free Red Bull® were cytotoxic in HL-60 cell line. Classic Red Bull® induced DNA internucleosomal fragmentation although none of them exhibited DNA damage on human leukaemia cells. In conclusion, the tested compounds are safe on Drosophila melanogaster and classic Red Bull® could overall possess nutraceutical potential in the in vivo and in vitro model used in this study. Besides, taurine could holistically be one of the bioactive compounds responsible for the biological activity of classic Red Bull®.

scholarly journals Influence of Dopamine on Fluorescent Advanced Glycation End Products Formation Using Drosophila melanogaster

Biomolecules ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 453
Author(s):  
Ana Filošević Vujnović ◽  
Katarina Jović ◽  
Emanuel Pištan ◽  
Rozi Andretić Waldowski
Keyword(s):  
Drosophila Melanogaster ◽  
Advanced Glycation End Products ◽  
Model Organism ◽  
Covalent Modification ◽  
Advanced Glycation ◽  
Biomarkers Of Aging ◽  
Glycation End Products ◽  
End Products

Non-enzymatic glycation and covalent modification of proteins leads to Advanced Glycation End products (AGEs). AGEs are biomarkers of aging and neurodegenerative disease, and can be induced by impaired neuronal signaling. The objective of this study was to investigate if manipulation of dopamine (DA) in vitro using the model protein, bovine serum albumin (BSA), and in vivo using the model organism Drosophila melanogaster, influences fluorescent AGEs (fAGEs) formation as an indicator of dopamine-induced oxidation events. DA inhibited fAGEs-BSA synthesis in vitro, suggesting an anti-oxidative effect, which was not observed when flies were fed DA. Feeding flies cocaine and methamphetamine led to increased fAGEs formation. Mutants lacking the dopaminergic transporter or the D1-type showed further elevation of fAGEs accumulation, indicating that the long-term perturbation in DA function leads to higher production of fAGEs. To confirm that DA has oxidative properties in vivo, we fed flies antioxidant quercetin (QUE) together with methamphetamine. QUE significantly decreased methamphetamine-induced fAGEs formation suggesting that the perturbation of DA function in vivo leads to increased oxidation. These findings present arguments for the use of fAGEs as a biomarker of DA-associated neurodegenerative changes and for assessment of antioxidant interventions such as QUE treatment.

scholarly journals PRESENCE OF A BASE LINE LEVEL OF SISTER CHROMATID EXCHANGES IN SOMATIC CELLS OF DROSOPHILA MELANOGASTER IN VIVO AND IN VITRO

Genetics ◽  
1982 ◽  
Vol 100 (2) ◽  
pp. 259-278
Author(s):  
Hideo Tsuji
Keyword(s):  
Drosophila Melanogaster ◽  
Sister Chromatid ◽  
Ganglion Cells ◽  
Sister Chromatid Exchanges ◽  
Base Line ◽  
Cell Cycles ◽  
Chromatid Exchanges ◽  
Third Instar Larvae

ABSTRACT Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2′-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 vg/ml and 0.25-2.5 vg/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).

scholarly journals Phosphorylation of Serine 303 Is a Prerequisite for the Stress-Inducible SUMO Modification of Heat Shock Factor 1

2003 ◽  
Vol 23 (8) ◽  
pp. 2953-2968 ◽  
Author(s):  
Ville Hietakangas ◽  
Johanna K. Ahlskog ◽  
Annika M. Jakobsson ◽  
Maria Hellesuo ◽  
Niko M. Sahlberg ◽  
...  
Keyword(s):  
Heat Shock ◽  
Phosphorylation Site ◽  
Stress Granules ◽  
Heat Shock Factor ◽  
Transcriptional Level ◽  
Heat Shock Factor 1 ◽  
Protection Mechanism ◽  
Sumo Modification

ABSTRACT The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylation-deficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.

Genetic analysis of oxygen defense mechanisms in Drosophila melanogaster and identification of a novel behavioural mutant with a Shaker phenotype

Genome ◽  
1996 ◽  
Vol 39 (4) ◽  
pp. 749-757 ◽  
Author(s):  
James M. Humphreys ◽  
Brenda Duyf ◽  
Mei-Ling A. Joiner ◽  
John P. Phillips ◽  
Arthur J. Hilliker
Keyword(s):  
Drosophila Melanogaster ◽  
Defense Mechanisms ◽  
Chromosome Region ◽  
Recessive Mutation ◽  
Ether Anaesthesia ◽  
Ros Metabolism ◽  
Chromosome Mutations ◽  
New Gene ◽  
New Alleles

Mutants of Drosophila melanogaster that lack Cu/Zn superoxide dismutase or urate are hypersensitive to reactive oxygen species (ROS) generated in vivo by the redox-cycling agent paraquat. We have subsequently employed paraquat as a selective agent to identify adult viable mutants potentially defective in other, perhaps unknown, components of ROS metabolism. Paraquat screening of ethyl methanesulfonate-induced second- and third-chromosome mutations yielded 24 paraquat hypersensitive mutants. Two mutants were identified as being new alleles of the previously identified doublesex (dsx) and pink (p) genes. The remainder of the mutations identified previously undescribed genes, including one second chromosome paraquat hypersensitive mutant that was found to exhibit shaking legs, abdomen pulsations, and body shuddering under ether anaesthesia. This recessive mutation was mapped to the polytene chromosome region of 48A5–48B2 and defines a new gene we named quiver (qvr). This mutation is similar in phenotype to the Shaker (Sh), ether-a-gogo (eag), and Hyperkinetic (Hk) mutations, all of which affect potassium channel function in D. melanogaster. Key words : Drosophila, paraquat, EMS-mutagenesis, Shaker, oxidative-stress.

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