29 research outputs found
Le système d expression du gène chloroplastique petA chez Chlamydomonas reinhardtii
No full textDepuis l endosymbiose ayant mené au chloroplaste de l algue verte unicellulaire Chlamydomonas reinhardtii, la plupart des gènes de l endosymbiote primitif ont été transférés dans le génome nucléaire de l hôte, qui code donc des sous-unités des complexes de l appareil photosynthétique, mais aussi des facteurs régulant l expression des gènes restés dans le chloroplaste. Le gène chloroplastique petA, codant le cytochrome f, possède deux facteurs spécifiques : MCA1 est responsable de la stabilisation de l ARNm petA, et TCA1 de l activation de sa traduction. D autre part, la synthèse du cytochrome f est réprimée quand celui-ci ne peut s assembler au sein du complexe b6f, par le contrôle par épistasie de synthèse (ou CES). Le mécanisme moléculaire permettant l expression du gène petA et son contrôle par le CES a été largement dévoilé grâce aux travaux présentés dans ce manuscrit : les facteurs MCA1 et TCA1 forment différents complexes de haut poids moléculaires, pouvant contenir l ARNm petA, et le cytochrome f non assemblé signalise la dégradation de MCA1 par les protéases FtsH et ClpP.Par ailleurs, ces travaux ont fortuitement mené à la découverte d une mutation nucléaire dominante, su0, conduisant à la dégradation de l ARNm petA, mais seulement lors de sa traduction. La mutation causant ce phénotype intriguant, indépendante des facteurs MCA1 et TCA1 et du CES, a été localisée dans une région de 300 kb sur le chromosome 15, mais n a pas encore été identifiée.PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF
The diurnal logic of the expression of the chloroplast genome in Chlamydomonas reinhardtii.
No full textChloroplasts are derived from cyanobacteria and have retained a bacterial-type genome and gene expression machinery. The chloroplast genome encodes many of the core components of the photosynthetic apparatus in the thylakoid membranes. To avoid photooxidative damage and production of harmful reactive oxygen species (ROS) by incompletely assembled thylakoid protein complexes, chloroplast gene expression must be tightly regulated and co-ordinated with gene expression in the nucleus. Little is known about the control of chloroplast gene expression at the genome-wide level in response to internal rhythms and external cues. To obtain a comprehensive picture of organelle transcript levels in the unicellular model alga Chlamydomonas reinhardtii in diurnal conditions, a qRT-PCR platform was developed and used to quantify 68 chloroplast, 21 mitochondrial as well as 71 nuclear transcripts in cells grown in highly controlled 12 h light/12 h dark cycles. Interestingly, in anticipation of dusk, chloroplast transcripts from genes involved in transcription reached peak levels first, followed by transcripts from genes involved in translation, and finally photosynthesis gene transcripts. This pattern matches perfectly the theoretical demands of a cell "waking up" from the night. A similar trend was observed in the nuclear transcripts. These results suggest a striking internal logic in the expression of the chloroplast genome and a previously unappreciated complexity in the regulation of chloroplast genes
The Nucleus-Encoded trans -Acting Factor MCA1 Plays a Critical Role in the Regulation of Cytochrome f Synthesis in Chlamydomonas Chloroplasts
No full textInternational audienc
Human MAIT cell cytolytic effector proteins synergize to overcome carbapenem resistance in Escherichia coli
No full textCopyright: © 2020 Boulouis et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Mucosa-associated invariant T (MAIT) cells are abundant antimicrobial T cells in humans and recognize antigens derived from the microbial riboflavin biosynthetic pathway presented by the MHC-Ib-related protein (MR1). However, the mechanisms responsible for MAIT cell antimicrobial activity are not fully understood, and the efficacy of these mechanisms against antibiotic resistant bacteria has not been explored. Here, we show that MAIT cells mediate MR1-restricted antimicrobial activity against Escherichia coli clinical strains in a manner dependent on the activity of cytolytic proteins but independent of production of pro-inflammatory cytokines or induction of apoptosis in infected cells. The combined action of the pore-forming antimicrobial protein granulysin and the serine protease granzyme B released in response to T cell receptor (TCR)-mediated recognition of MR1-presented antigen is essential to mediate control against both cell-associated and free-living, extracellular forms of E. coli. Furthermore, MAIT cell-mediated bacterial control extends to multidrug-resistant E. coli primary clinical isolates additionally resistant to carbapenems, a class of last resort antibiotics. Notably, high levels of granulysin and granzyme B in the MAIT cell secretomes directly damage bacterial cells by increasing their permeability, rendering initially resistant E. coli susceptible to the bactericidal activity of carbapenems. These findings define the role of cytolytic effector proteins in MAIT cell-mediated antimicrobial activity and indicate that granulysin and granzyme B synergize to restore carbapenem bactericidal activity and overcome carbapenem resistance in E. coli.</div
Confirmation of the diurnal expression patterns observed in the pRT-PCR experiments by northern blot analyses.
No full text<p>To avoid variation from RNA loading, each membrane was hybridized and stripped multiple times. The order of probing corresponds to the vertical order of the images (and the increasing signal intensity known to be obtained with the different probes). The ethidium bromide fluorescence (EtBr) remaining on the membrane after blotting is shown as a loading control.</p
Diurnal transcriptomics on bioreactor cultures of <i>Chlamydomonas</i>.
No full text<p>The effect of 12 h light/12 h dark cycles on organelle (and selected nuclear) transcripts was analyzed by qRT-PCR. <i>C. reinhardtii</i> cells were grown in six independent bioreactor runs (run numbers 1, 2, 3, 15, 16, 20) and harvested at the time points indicated. Data obtained for plastome transcripts is shown in the upper portion of the heatmap, chondriome transcripts in the central region, and nuclear transcripts in the lower region. Within each genome, transcripts are listed alphabetically. Data are normalized to housekeeping transcripts and then to the average across all samples for that gene. Red boxes indicate up-regulation, green boxes: down-regulation, grey boxes: no data. Visualization by Multiexperiment Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108760#pone.0108760-Saeed1" target="_blank">[38]</a>.</p
Behavior of functionally related chloroplast transcripts in diurnal conditions.
No full text<p>The average peak time of all transcripts belonging to the three functional classes (red: plastid-encoded RNA polymerase genes, PEP; blue: ribosome-related genes; green: photosynthesis-related genes) is shown for each bioreactor experiment. The intensity of the circle represents the number of transcripts peaking at the time, and the black x represents the average of all transcripts. The yellow bar represents the 12 h light period, flanked by dark periods (grey bars).</p
Key bioreactor parameters from a representative bioreactor run (R2) in diurnal conditions.
No full text<p>Turbidity (navy blue) slightly decreased during the dark period and increased during the light. The rapid peak after dusk and rapid drop after dawn are technical artefacts of the turbidimeter. The peaks in the weight graph correspond to the sampling times, when the fermenter was disturbed. Dissolved oxygen concentration increases from ca. 80% to ca. 150% very rapidly in the light. The pH of the culture is controlled by titration of acid or base, temperature is controlled by dynamic cooling to maintain reasonably stable values for these parameters. Samples were taken every 4 h, these points are indicated and also visible as peaks in the weight data (purple). Yellow bars represents the 12 h light period (L), flanked by dark periods (D; grey bars).</p
Comparison of microarray and qRT-PCR-based quantification of transcripts in a diurnal rhythm.
No full text<p>Changes in organelle (chloroplast and mitochondrial) transcript levels between time points L2 and D2 in a wild-type strain of <i>C. reinhardtii</i> (CC-124) were determined by microarray and by qRT-PCR. The resulting fold-changes correlate with an R<sup>2</sup> value of 0.66. It is lower than 1.0, mainly because the microarray produces less accurate results for lowly expressed genes. All data points represent the average of three independent experiments.</p
k-means clustering of diurnal qRT-PCR data for chloroplast transcripts (left) and a subset of nuclear transcripts (right).
No full text<p>The averaged data for six biological replicates were grouped into three clusters with the Pearson correlation as the distance metric. Gene profiles are colored according to the gene product’s function. Green: photosynthesis-related genes, blue: ribosome-related genes, red: plastid-encoded RNA polymerase genes, grey: miscellaneous genes. Clustering and visualization by Multiexperiment Viewer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108760#pone.0108760-Saeed1" target="_blank">[38]</a>.</p
