18 results on '"Clara, Moch"'
Search Results
2. Translational regulation by RACK1 in astrocytes represses KIR4.1 expression and regulates neuronal activity
- Author
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Marc Oudart, Katia Avila-Gutierrez, Clara Moch, Elena Dossi, Giampaolo Milior, Anne-Cécile Boulay, Mathis Gaudey, Julien Moulard, Bérangère Lombard, Damarys Loew, Alexis-Pierre Bemelmans, Nathalie Rouach, Clément Chapat, and Martine Cohen-Salmon
- Abstract
SummaryThe regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomic screen for polysome-associated proteins in astrocytes and focused on the ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially bound to a number of mRNAs, including Kcnj10, encoding the inward rectifying potassium (K+) channel KIR4.1, a critical astrocytic regulator of neurotransmission. By developing an astrocyte-specific, conditional RACK1 knock-out mouse model, we showed that RACK1 repressed the production of KIR4.1 in hippocampal astrocytes and PAPs. Reporter-based assays revealed that RACK1 controlled Kcnj10 translation through the transcript’s 5’ untranslated region. Upregulation of KIR4.1 in the absence of RACK1 modified the astrocyte territory volume and neuronal activity attenuatin burst frequency and duration in the hippocampus. Hence, astrocytic RACK1 represses KIR4.1 translation and influences neuronal activity.
- Published
- 2022
3. The SARS-CoV-2 protein NSP2 impairs the silencing capacity of the human 4EHP-GIGYF2 complex
- Author
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Limei Zou, Clara Moch, Marc Graille, and Clément Chapat
- Subjects
Multidisciplinary - Abstract
There is an urgent need for a molecular understanding of how SARS-CoV-2 influences the machineries of the host cell. Herein, we focused our attention on the capacity of the SARS-CoV-2 protein NSP2 to bind the human 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression. Using
- Published
- 2022
4. The SARS-CoV-2 protein NSP2 impairs the microRNA-induced silencing capacity of human cells
- Author
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Limei Zou, Clara Moch, Marc Graille, and Clément Chapat
- Subjects
viruses ,virus diseases ,biochemical phenomena, metabolism, and nutrition - Abstract
The coronavirus SARS-CoV-2 is the cause of the ongoing pandemic of COVID-19. Given the absence of effective treatments against SARS-CoV-2, there is an urgent need for a molecular understanding of how the virus influences the machineries of the host cell. The SARS-CoV-2 generates 16 Non-Structural Proteins (NSPs) through proteolytic cleavage of a large precursor protein. In the present study, we focused our attention on the SARS-CoV-2 protein NSP2, whose role in the viral pathogenicity is poorly understood. Recent proteomic studies shed light on the capacity of NSP2 to bind the 4EHP-GIGYF2 complex, a key factor involved in microRNA-mediated silencing of gene expression in human cells. In order to gain a better understanding of the function of NSP2, we attempted to identify the molecular basis of its interaction with 4EHP-GIGYF2. Our data demonstrate that NSP2 physically associates with the endogenous 4EHP-GIGYF2 complex in the cytoplasm. Using co-immunoprecipitation and in vitro interaction assays, we identified both 4EHP and a central segment in GIGYF2 as binding sites for NSP2. We also provide functional evidence that NSP2 impairs the function of GIGYF2 in mediating mRNA silencing using reporter-based assays, thus leading to a reduced activity of microRNAs. Altogether, these data reveal the profound impact of NSP2 on the post-transcriptional silencing of gene expression in human cells, pointing out 4EHP-GIGYF2 targeting as a possible strategy of SARS-CoV-2 to take over the silencing machinery and to suppress host defenses.
- Published
- 2022
5. Spermidine strongly increases the fidelity of Escherichia coli CRISPR Cas1–Cas2 integrase
- Author
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Clara Moch, Sylvain Blanquet, and Pierre Plateau
- Subjects
0301 basic medicine ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Cell Biology ,medicine.disease_cause ,Biochemistry ,Integrase ,Cell biology ,Spermidine ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Plasmid ,biology.protein ,medicine ,CRISPR ,Protein–DNA interaction ,Polyamine ,Molecular Biology ,Escherichia coli ,DNA - Abstract
Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1-Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro However, this nonspecific integration activity is rare in vivo Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1-Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1-Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.
- Published
- 2019
6. Spermidine strongly increases the fidelity of
- Author
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Pierre, Plateau, Clara, Moch, and Sylvain, Blanquet
- Subjects
DNA, Bacterial ,Integration Host Factors ,Binding Sites ,Integrases ,DNA, Superhelical ,Spermidine ,Escherichia coli Proteins ,Escherichia coli ,Enzymology ,CRISPR-Cas Systems - Abstract
Site-selective CRISPR array expansion at the origin of bacterial adaptive immunity relies on recognition of sequence-dependent DNA structures by the conserved Cas1–Cas2 integrase. Off-target integration of a new spacer sequence outside canonical CRISPR arrays has been described in vitro. However, this nonspecific integration activity is rare in vivo. Here, we designed gel assays to monitor fluorescently labeled protospacer insertion in a supercoiled 3-kb plasmid harboring a minimal CRISPR locus derived from the Escherichia coli type I-E system. This assay enabled us to distinguish and quantify target and off-target insertion events catalyzed by E. coli Cas1–Cas2 integrase. We show that addition of the ubiquitous polyamine spermidine or of another polyamine, spermine, significantly alters the ratio between target and off-target insertions. Notably, addition of 2 mm spermidine quenched the off-target spacer insertion rate by a factor of 20-fold, and, in the presence of integration host factor, spermidine also increased insertion at the CRISPR locus 1.5-fold. The observation made in our in vitro system that spermidine strongly decreases nonspecific activity of Cas1–Cas2 integrase outside the leader-proximal region of a CRISPR array suggests that this polyamine plays a potential role in the fidelity of the spacer integration also in vivo.
- Published
- 2019
7. Redesigning the stereospecificity of tyrosyl-tRNA synthetase
- Author
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Clara Moch, Pierre Plateau, Thomas Simonson, Karen Druart, Najette Amara, Erwan Bigan, Zoltan Palmai, Shixin Ye-Lehmann, and Sandra Wydau-Dematteis
- Subjects
0301 basic medicine ,biology ,Chemistry ,Stereochemistry ,Protein design ,Active site ,Protein engineering ,010402 general chemistry ,Genetic code ,Ligand (biochemistry) ,01 natural sciences ,Biochemistry ,0104 chemical sciences ,03 medical and health sciences ,030104 developmental biology ,Stereospecificity ,Tyrosine—tRNA ligase ,Structural Biology ,Transfer RNA ,biology.protein ,Molecular Biology - Abstract
D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.
- Published
- 2016
8. DNA binding specificities of Escherichia coli Cas1–Cas2 integrase drive its recruitment at the CRISPR locus
- Author
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Clara, Moch, Michel, Fromant, Sylvain, Blanquet, Pierre, Plateau, Laboratoire de Biochimie de l'Ecole polytechnique (BIOC), and École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
DNA, Bacterial ,MESH: Endodeoxyribonucleases/metabolism ,MESH: Plasmids/metabolism ,CRISPR-Associated Proteins ,MESH: Endonucleases/metabolism ,MESH: Binding, Competitive ,MESH: Escherichia coli/genetics ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Binding, Competitive ,MESH: Escherichia coli Proteins/metabolism ,MESH: DNA, Bacterial/metabolism ,Escherichia coli ,MESH: Protein Binding ,Clustered Regularly Interspaced Short Palindromic Repeats ,Endodeoxyribonucleases ,Integrases ,Nucleic Acid Enzymes ,Escherichia coli Proteins ,Inverted Repeat Sequences ,MESH: CRISPR-Associated Proteins/metabolism ,[SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology ,Endonucleases ,[SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM] ,MESH: Integrases/metabolism ,MESH: Clustered Regularly Interspaced Short Palindromic Repeats ,MESH: Inverted Repeat Sequences ,Plasmids ,Protein Binding - Abstract
International audience; Prokaryotic adaptive immunity relies on the capture of fragments of invader DNA (protospacers) followed by their recombination at a dedicated acceptor DNA locus. This integrative mechanism, called adaptation, needs both Cas1 and Cas2 proteins. Here, we studied in vitro the binding of an Escherichia coli Cas1-Cas2 complex to various protospacer and acceptor DNA molecules. We show that, to form a long-lived ternary complex containing Cas1-Cas2, the acceptor DNA must carry a CRISPR locus, and the protospacer must not contain 3΄-single-stranded overhangs longer than 5 bases. In addition, the acceptor DNA must be supercoiled. Formation of the ternary complex is synergistic, in such that the binding of Cas1-Cas2 to acceptor DNA is reinforced in the presence of a protospacer. Mutagenesis analysis at the CRISPR locus indicates that the presence in the acceptor plasmid of the palindromic motif found in CRISPR repeats drives stable ternary complex formation. Most of the mutations in this motif are deleterious even if they do not prevent cruciform structure formation. The leader sequence of the CRISPR locus is fully dispensable. These DNA binding specificities of the Cas1-Cas2 integrase are likely to play a major role in the recruitment of this enzyme at the CRISPR locus.
- Published
- 2017
9. Live-imaging of single stem cells within their niche reveals that a U3snoRNP component segregates asymmetrically and is required for self-renewal in Drosophila
- Author
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Clara M Sidor, Jean-René Huynh, Charlotte Martin, Kenzo Ivanovitch, Yohanns Bellaïche, Pierre Fichelson, Jean-Antoine Lepesant, Clara Moch, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Medical Research Council, Laboratory for Molecular Cell Biology and Cell Biology Unit, Department of Anatomy and Developmental Biology,University College London, University College of London [London] (UCL), Génétique du Développement et Cancer, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Cancer Research UK London Research Institute, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC)
- Subjects
Recombinant Fusion Proteins ,Niche ,[SDV.BC]Life Sciences [q-bio]/Cellular Biology ,Self renewal ,Biology ,Ribosome ,03 medical and health sciences ,Ribonucleoproteins, Small Nucleolar ,Live cell imaging ,Component (UML) ,RNA Precursors ,Animals ,Drosophila Proteins ,[SDV.BDD]Life Sciences [q-bio]/Development Biology ,030304 developmental biology ,Neurons ,0303 health sciences ,Stem Cells ,fungi ,030302 biochemistry & molecular biology ,RNA ,Cell Biology ,Cell biology ,Drosophila melanogaster ,Oocytes ,RNA Interference ,Stem cell ,Function (biology) ,Signal Transduction - Abstract
International audience; Stem cells generate self-renewing and differentiating progeny over many rounds of asymmetric divisions. How stem cell growth rate and size are maintained over time remains unknown. We isolated mutations in a Drosophila melanogaster gene, wicked (wcd), which induce premature differentiation of germline stem cells (GSCs). Wcd is a member of the U3 snoRNP complex required for pre-ribosomal RNA maturation. This general function of Wcd contrasts with its specific requirement for GSC self-renewal. However, live imaging of GSCs within their niche revealed a pool of Wcd-forming particles that segregate asymmetrically into the GSCs on mitosis, independently of the Dpp signal sent by the niche. A fraction of Wcd also segregated asymmetrically in dividing larval neural stem cells (NSCs). In the absence of Wcd, NSCs became smaller and produced fewer neurons. Our results show that regulation of ribosome synthesis is a crucial parameter for stem cell maintenance and function.
- Published
- 2009
10. Redesigning the stereospecificity of tyrosyl-tRNA synthetase
- Author
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Thomas, Simonson, Shixin, Ye-Lehmann, Zoltan, Palmai, Najette, Amara, Sandra, Wydau-Dematteis, Erwan, Bigan, Karen, Druart, Clara, Moch, and Pierre, Plateau
- Subjects
Tyrosine-tRNA Ligase ,Escherichia coli Proteins ,Thermodynamics ,Stereoisomerism ,Molecular Dynamics Simulation ,Protein Engineering - Abstract
D-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled.
- Published
- 2015
11. Gene structure of the ciliate Sterkiella histriomuscorum based on a combined analysis of DNA and cDNA sequences from 21 macronuclear chromosomes
- Author
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Clara Moch, Tie Yang, Loïc Morin, Rachel Lescasse, Eduardo Villalobo, J. Grisvard, and Anne Baroin-Tourancheau
- Subjects
DNA, Complementary ,Molecular Sequence Data ,Gene Expression ,Biology ,Chromosomes ,Conserved sequence ,chemistry.chemical_compound ,Minichromosome ,Complementary DNA ,Gene Order ,Genetics ,Animals ,Coding region ,Ciliophora ,3' Untranslated Regions ,Gene ,Genetics (clinical) ,Base Sequence ,Macronucleus ,Intron ,DNA ,Introns ,Blotting, Southern ,chemistry ,Transcription Initiation Site ,5' Untranslated Regions - Abstract
Macronuclear deoxyribonucleic acid (DNA) in hypotrichous ciliates consists of a set of linear molecules ranging in size from 0.5 to several tens of kilobases and typically carrying a single gene. Each minichromosome is present at a ploidy ofor=1,000 per macronucleus. These molecules are known as gene-sized molecules. Multigene molecules are also present, but are still poorly described. In analyzing the encystment-excystment cycle of Sterkiella histriomuscorum, we have characterized a set of 21 macronuclear molecules both at the DNA and complementary DNA (cDNA) levels. On a total of 23 validated coding sequences, we mapped the 5' and 3' untranslated regions for a subset of 10 and 18 transcripts, respectively. A combination of DNA and cDNA data allows us to precisely determine several structural features of macronuclear chromosomes, such as the organization of multigene molecules, an intron content higher than expected, and a conserved sequence surrounding the initiation transcription site. It also reveals one coding sequence containing a transcribed 10-bp element that displays the characteristic features of internal eliminated sequences (IES). Its presence in a fraction of the minichromosomes carrying this gene raises the possibility of an incomplete IES excision process during the development of the S. histriomuscorum macronucleus.
- Published
- 2005
12. Cysteine Proteases and Cell Differentiation: Excystment of the Ciliated Protist Sterkiella histriomuscorum
- Author
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Loïc Morin, Ghislaine Fryd-Versavel, Eduardo Villalobo, Anne Fleury-Aubusson, Clara Moch, and Universidad de Sevilla. Departamento de Microbiología
- Subjects
Proteases ,Time Factors ,Leupeptins ,Genes, Protozoan ,Molecular Sequence Data ,Protozoan Proteins ,Cysteine Proteinase Inhibitors ,Biology ,Microbiology ,Article ,Cathepsin B ,Conserved sequence ,chemistry.chemical_compound ,Cell Wall ,Cathepsin H ,Animals ,Amino Acid Sequence ,Ciliophora ,Molecular Biology ,Peptide sequence ,Conserved Sequence ,Phylogeny ,Cathepsin ,Dose-Response Relationship, Drug ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,Leupeptin ,Cell Differentiation ,Calpain ,Dipeptides ,General Medicine ,Protein Structure, Tertiary ,Cysteine Endopeptidases ,Biochemistry ,chemistry ,biology.protein - Abstract
The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.
- Published
- 2003
13. P16 UV mutations in human skin epithelial tumors
- Author
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Jacqueline Rivet, C. Vilmer, Louis Dubertret, Anne Tesniere, Clara Moch, Jean-Pierre Molès, Nicole Basset-Seguin, Olivier Verola, and Nadem Soufir
- Subjects
Cancer Research ,Neoplasms, Radiation-Induced ,Skin Neoplasms ,Tumor suppressor gene ,Ultraviolet Rays ,Gene mutation ,Biology ,medicine.disease_cause ,Exon ,Proto-Oncogene Proteins ,Tumor Suppressor Protein p14ARF ,Genetics ,medicine ,Humans ,Basal cell carcinoma ,Neoplasms, Glandular and Epithelial ,Molecular Biology ,Cyclin-Dependent Kinase Inhibitor p16 ,Mutation ,Actinic keratosis ,Cyclin-Dependent Kinase 4 ,Proteins ,Exons ,medicine.disease ,Cyclin-Dependent Kinases ,Introns ,Alternative Splicing ,Epidermoid carcinoma ,Carcinoma, Basal Cell ,Carcinoma, Squamous Cell ,Cancer research ,Tumor Suppressor Protein p53 ,Carcinogenesis - Abstract
The p16 gene expresses two alternative transcripts (p16alpha and p16beta) involved in tumor suppression via the retinoblastoma (Rb) or p53 pathways. Disruption of these pathways can occur through inactivation of p16 or p53, or activating mutations of cyclin dependant kinase 4 gene (Cdk4). We searched for p16, Cdk4 and p53 gene mutations in 20 squamous cell carcinomas (SSCs), 1 actinic keratosis (AK), and 28 basal cell carcinomas (BCCs), using PCR-SSCP. A deletion and methylation analysis of p16 was also performed. Six different mutations (12%) were detected in exon 2 of p16 (common to p16alpha and p16beta), in five out of 21 squamous lesions (24%) (one AK and four SCCs) and one out of 28 BCCs (3.5%). These included four (66%) ultraviolet (UV)-type mutations (two tandems CC : GG to TT : AA transitions and two C : G to T : A transitions at dipyrimidic site) and two transversions. P53 mutations were present in 18 samples (37%), mostly of UV type. Of these, only two (one BCC and one AK) harboured simultaneously mutations of p16, but with no consequence on p16beta transcript. Our data demonstrate for the first time the presence of p16 UV induced mutations in non melanoma skin cancer, particularly in the most aggressive SCC type, and support that p16 and p53 are involved in two independent pathways in skin carcinogenesis.
- Published
- 1999
14. [Untitled]
- Author
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Dominique Daegelen, Axel Kahn, Arlette Porteu, François Spitz, and Clara Moch
- Subjects
Genetically modified mouse ,Reporter gene ,biology ,Transgene ,Aldolase A ,Fructose-bisphosphate aldolase ,Promoter ,Molecular biology ,Chromatin ,Gene expression ,Genetics ,biology.protein ,Animal Science and Zoology ,Agronomy and Crop Science ,Biotechnology - Abstract
In order to identify regulatory elements that direct widespread in vivo expression of a linked gene, we have examined one of the human aldolase A alternative promoters, the ubiquitous pH promoter, which is active in most foetal and adult tissues. We have used the pH promoter region to drive expression of an heterologous CAT reporter gene in transgenic mice. We show that a short 820 bp pH promoter fragment is able to confer a ubiquitous and reproducible activity pattern on the CAT reporter gene in most of the transgenic lines analysed, with a particularly high level of expression in adult skeletal muscle. Activity of this transgene was detected from early embryonic stages. Therefore, this pH promoter region appears to be a powerful tool to direct ubiquitous and early expression of a transgene in vivo. Deletion analysis revealed that: (i) the region between −651 and −369 bp relative to the pH promoter transcription start site includes DNA elements capable of overriding effects of the surrounding chromatin at the integration site, (ii) the region between −285 and −211 bp is involved in pH promoter tissue-specific expression pattern in skeletal muscle and/or nervous tissues, (iii) the region located between −211 and −108 bp is necessary for its ubiquitous and muscle-pre dominant activity and (iv) the most proximal region downstream from −108 bp is still sufficient to confer an activity in brain and lung
- Published
- 1998
15. A homologue of CROC-1 in a ciliated protist (Sterkiella histriomuscorum) testifies to the ancient origin of the ubiquitin-conjugating enzyme variant family
- Author
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Loïc Morin, Rachel Lescasse, Eduardo Villalobo, Anne Baroin-Tourancheau, Wei Xiao, Clara Moch, and Michelle Hanna
- Subjects
Saccharomyces cerevisiae ,Protozoan Proteins ,Ubiquitin-conjugating enzyme ,Evolution, Molecular ,Ligases ,chemistry.chemical_compound ,Structure-Activity Relationship ,Genetics ,Animals ,Cluster Analysis ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Ciliophora ,Cloning, Molecular ,Molecular Biology ,Gene ,Ecology, Evolution, Behavior and Systematics ,Conserved Sequence ,Phylogeny ,Regulator gene ,Regulation of gene expression ,Messenger RNA ,biology ,Phylogenetic tree ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,Ubiquitin ,Nuclear Proteins ,biology.organism_classification ,Introns ,chemistry ,Ubiquitin-Conjugating Enzymes ,Trans-Activators ,Transcription Initiation Site ,DNA ,Transcription Factors - Abstract
Resting cysts of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) have been shown to contain messenger RNA, one of which codes for a protein significantly similar to CROC-1. CROC-1 is a human regulatory protein capable of transactivating the promoter of c-fos and belongs to a newly characterized family of ubiquitin-conjugating enzyme (E2) variants (UEV). We have determined the corresponding macronuclear gene sequence, which is the first protistan UEV sequence available. The phylogenetic analysis indicates the deep separation and solid clustering of all the UEV sequences within the E2 tree showing the ancient origin of these regulatory genes and their high structural conservation during evolution. Furthermore, overexpression of the ciliate UEV is able to rescue the Saccharomyces cerevisiae mms2 null mutant from killing by DNA damaging agents, implying that the UEV family proteins are functionally conserved. In S. histriomuscorum, expression of UEV is correlated with the growth of the cells as transcripts are present in excysting and vegetative cells but are rapidly down-regulated during starvation. These data support the high conservation of the UEV family in eukaryotes, and a regulatory role of the gene is discussed in relation to known functions of UEVs. This analysis may promote the search for homologues of other regulatory genes (metazoan regulators of differentiation) in ciliates.
- Published
- 2001
16. Searching for excystment-regulated genes in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): a mRNA differential display analysis of gene expression in excysting cells
- Author
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Eduardo Villalobo, Anne Baroin-Tourancheau, Roland Perasso, and Clara Moch
- Subjects
Oxytrichidae ,Somatic cell ,Molecular Sequence Data ,Biology ,Oxytricha ,Microbiology ,Gene expression ,Animals ,Northern blot ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Gene ,Differential display ,Base Sequence ,Sequence Homology, Amino Acid ,Reverse Transcriptase Polymerase Chain Reaction ,Inverse polymerase chain reaction ,Gene Expression Profiling ,Gene Expression Regulation, Developmental ,DNA, Protozoan ,biology.organism_classification ,Blotting, Northern ,Molecular biology ,Blotting, Southern - Abstract
In the absence of food, the oxytrichid Sterkiella histriomuscorum transforms like many ciliates into resting cysts. When transferred back into feeding medium, the cyst re-transforms into a vegetative cell. The entry into and exit from the dormant cyst stage are complex developmental processes still poorly investigated at the molecular level. Assuming that these changes in state could involve changes in gene expression, we have used the technique of mRNA differential display to detect differentially expressed genes in cysts and two different stages of excysting cell. Variation in the temporal expression pattern of transcripts could be detected and, in using an inverse-PCR strategy on circularized macronuclear DNA, we have sequenced the macronuclear genes of three of the isolated cDNAs, which correspond to 1) a nucleotide-binding domain-encoding gene, 2) a DHHC-domain-carrying gene, and 3) a phosphatase type 2C-encoding gene. For the first two genes, Northern blot analyses supported an excystment-associated regulated gene expression. We discuss their possible role during excystment and we show that the combination of differential display and inverse PCR constitutes a powerful approach to isolate excystment-regulated genes in hypotrichs.
- Published
- 2001
17. An opportunistic promoter sharing regulatory sequences with either a muscle-specific or a ubiquitous promoter in the human aldolase A gene
- Author
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Pascal Maire, Josiane Demignon, Marjo Salminen, Axel Kahn, Jean-Paul Concordet, Dominique Daegelen, and Clara Moch
- Subjects
DNA, Recombinant ,Mice, Transgenic ,Biology ,Gene Expression Regulation, Enzymologic ,Mice ,Fructose-Bisphosphate Aldolase ,Gene expression ,medicine ,Animals ,Humans ,RNA, Messenger ,Enhancer ,Promoter Regions, Genetic ,Gene ,Molecular Biology ,Locus control region ,Muscles ,Aldolase A ,Skeletal muscle ,Promoter ,Cell Differentiation ,Cell Biology ,Molecular biology ,Globins ,medicine.anatomical_structure ,Enhancer Elements, Genetic ,Regulatory sequence ,biology.protein ,Research Article - Abstract
The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.
- Published
- 1993
18. Fast-muscle-specific expression of human aldolase A transgenes
- Author
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A Kahn, Arlette Porteu, Clara Moch, Dominique Daegelen, Jean-Paul Concordet, Pascal Maire, and Marjo Salminen
- Subjects
Chloramphenicol O-Acetyltransferase ,Transcription, Genetic ,Transgene ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Fructose-bisphosphate aldolase ,Mice, Transgenic ,Mice ,Fructose-Bisphosphate Aldolase ,Animals ,Deoxyribonuclease I ,Humans ,Tissue Distribution ,RNA, Messenger ,Promoter Regions, Genetic ,Molecular Biology ,Regulation of gene expression ,biology ,Base Sequence ,Muscles ,Aldolase A ,Promoter ,Cell Biology ,Molecular biology ,Gene Expression Regulation ,Regulatory sequence ,Multigene Family ,biology.protein ,Hypersensitive site ,Research Article - Abstract
The expression of the human aldolase A gene is controlled by three alternative promoters. In transgenic mice, pN and pH are active in all tissues whereas pM is activated specifically in adult muscles composed mainly of fast, glycolytic fibers. To detect potential regulatory regions involved in the fast-muscle-specific activation of pM, we analyzed DNase I hypersensitivity in a 4.3-kbp fragment from the 5' end of the human aldolase A gene. Five hypersensitive sites were located near the transcription initiation site of each promoter in those transgenic-mouse tissues in which the corresponding promoter was active. Only one muscle-specific hypersensitive site was detected, mapping near pM. To functionally delimit the elements required for muscle-specific activity of pM, we performed a deletion analysis of the aldolase A 5' region in transgenic mice. Our results show that a 280-bp fragment containing 235 bp of pM proximal upstream sequences together with the noncoding M exon is sufficient for tissue-specific expression of pM. When a putative MEF-2-binding site residing in this proximal pM region is mutated, pM is still active and no change in its tissue specificity is detected. Furthermore, we observed a modulation of pM activity by elements lying further upstream and downstream from pM. Interestingly, pM was expressed in a tissue-specific way in all transgenic mice in which the 280-bp region was present (32 lines and six founder animals). This observation led us to suggest that the proximal pM region contains elements that are able to override to some extent the effects of the surrounding chromatin.
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