, La formation des manteaux COPII est régulée par le Sar1p-GTPase. L'attachement de Sar1p avec le RE requiert la liaison de GTP, processus facilité par la protéine d'échange de GTP (GEF) Sec12. Sar1p-GTP recrute les sous-complexes Sec23-Sec24p (bleu clair) du manteau COPII, ce qui permet le recrutement des protéines de cargo (vert clair), vers les sites de sorties du RE (ERES), et des protéines Sec13-Sec31p (violet), qui induisent la courbure de membrane et la formation de vésicules. L'hydrolyse du GTP de Sar1p par Sec23p a pour conséquence de désassembler le manteau COPII. La vésicule s'arrime ensuite aux membranes du ERGIC (ou cis-Golgi) par l'attachement de ses protéines, via les vésicules recouvertes de COPII
, La formation des manteaux de COPI est régulée par l'Arf1GTPase. L'interaction d'Arf1 avec l'ERGIC requiert la liaison de GTP, facilitée par les protéines GEF GBF1 ou BIG1/2. Arf1-GTP recrute les complexes de manteau de COPI (bleu foncé) du cytosol, provoquant la courbure des membranes et la formation de vésicules adressée au RE. L'hydrolyse d'Arf1-GTP, par une protéine activatrice d'Arf1-GTP (Arf-GAP), Golgi) vers le RE pour rapporter les protéines du Golgi et du ERGIC au RE, via les vésicules recouvertes de COPI, 2007.
µL de chloroforme : alcool Isoamylique (24 :1) est ajouté dans les tubes, vortexé 1 min et laissé à température ambiante 10 min. Les tubes sont centrifugés 15 min à 4°C 12000 g ,
, Les ARN sont dosés au Nanodrop et leur qualité est vérifiée sur un gel d'agarose. Les tubes d'ARN sont stockés à-80°C
, Quantification d'acides nucléiques
, Quantification rapide En routine, la quantification d'acides nucléiques est soit mesurée au nanodrop ND Spectrophotometer qui permet des mesures à 230, 260 et 280 nm
, De manière occasionnelle, nous avons employé la RT-qPCR
M0303S) : 500 ng d'ARN totaux sont traités, dans un volume total de 5 µl, pendant 30 min, à 37°C, par la DNase I. L'inactivation de l'enzyme est réalisée en ajoutant 0,5 µl d'EDTA (5 mM) et en portant les tubes 10 min à 65°C ,
, Fermentas) et de dNTP (5 mM). La RevertAid Premium Reverse Transcriptase (Thermo scientifique, #EP0733), son tampon (1X) ainsi que 10 unités de Ribolock RNase Inhibitor (Thermo scientifique, #E00382) sont ajoutés aux échantillons qui sont ensuite incubés 10 min à 25°C, Ces ARN sont placés 5 minutes à 65°C en présence d'amorces aléatoires, vol.100, p.30
, min à 50°C et enfin 5 min à 85°C. Les tubes contenant les cDNA sont stockés à-20°C
2010)), les amorces spécifiques de l'ARN viral, ainsi que la solution EvaGreen® (5X HOT Pol EvaGreen qPCR® Mix Plus (ROX), Euromedex) dans un volume total de 10 µl. Le thermocycleur qPCR LC480 (Roche) permet la réalisation de la qPCR, Le mélange réactionnel de qPCR contient 1,33 ng des cDNA, les amorces PDF2-5 et SAND-2 (permettant la quantification de deux gènes de référence ,
, , 2009.
, 1 unité par réaction) avec le tampon GC (Thermo F519) et avec des amorces permettant le recouvrement entre les extrémités des fragments sur au moins 25 pb. La dénaturation initiale est à 98°C (30 sec, pour la dénaturation initiale, puis 10 sec pour la dénaturation de chaque cycle), l'hybridation est dépendante du Tm des amorces (30 sec), l'élongation est réalisée à 72°C (30 sec par kb par cycle, Les fragments d'ADN à assembler sont amplifiés par PCR haute-fidélité par la Polymerase Phusion (Thermo F530S
, Les produits PCR sont déposés sur gels d'agarose pour la vérification et le prélèvement de la bande d'intérêt. Les fragments sont ensuite purifiés par un kit d'extraction sur gel d'agarose
, Au préalable , sont préparés 300 µl de mix AMM, qui comprennent l'Isothermal Reaction Buffer (IRB, concentré 5 fois, contenant 0,5 mg/µl de PEG 8000, des dNTPs à 100 mM et du NAD à 50 mM), vol.530, p.10
, en présence de MgCl2 (50 mM) et de DTT (100 mM). Ce mix est ensuite aliquoté par 15 µl et stocké à-20°C, la Taq DNA-ligase (NEB, M0208S, 1600 unités)
, Un mélange équimolaire des fragments à assembler est ajouté à un aliquot de mix AMM et l'ensemble est placé pendant 1 h à 50°C. La 5'-exonucléase est inactivée à 50°C en quelques minutes et n'élimine ainsi que quelques nucléotides en 5' des fragments d'ADN. Les régions de recouvrement des extrémités des fragments s'hybrident
, Cette technique a été employée pour générer les plasmides E17-stop69K avec les différents mutants (Figure M. 3). La liste de l'ensemble des plasmides cités se trouve en annexes
, Le culot est repris dans 10 ml de tampon I, centrifugé de nouveau (5min, 1500 g, 4°C) puis repris dans 10 ml de tampon I. La suspension bactérienne est laissée 1h dans la glace suivie d'une dernière centrifugation (5 min, 1500 g, 4°C). Le culot est repris dans 10 ml de tampon II
, MnCl2 45 mM, CaCl2 10 mM, HACoCl3 3 mM, KMes 10 mM, pH 6,3 via KOH, stérilisé par filtration sur 0, Tampon I KCl, vol.100
, RbCl 10 mM, CaCl2 75 mM, glycérol 15 % (v/v), pH 6,8 via NaOH, stérilisé par filtration sur 0, Tampon II MOPS 10 mM, vol.2
, Vérification des séquences d'ADN
, Les chromatogrammes des séquences sont vérifiés grâce au logiciel 4 peaks
, Les séquences nucléotidiques sont analysées à l'aide de Serial Cloner (version 2-6-1) Annexes
,
, Plasmide donneur mutant (dérivé p?-EGFP-140K)
, Plasmide receveur p?-CtYFP-98K
, La séquence mutée est encadrée par les sites de restriction : BamHI (site unique) et BstEII (site unique)
, Afin d'éliminer les fragments qui ne nous intéressent pas, les plasmides seront en plus digérés :-pour les plasmides donneurs : par BglI qui clive 2 fois en 864 et 7786.-pour les plasmides receveurs : par BsiWI qui clive une fois, Le plasmide receveur p?-CtYFP-98K possède ces mêmes sites
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