Nous remercions Wilkens Stephan à l&#39;aide de la microscopie électronique à transmission. Ce travail a été financé par les Instituts de Grant Santé R01AG023731 nationale.

Des études antérieures ont montré que les 22 premiers résidus amino-terminaux de Mgm101 sont clivés après l&#39;importation dans les mitochondries 19. Pour l&#39;expression chez Escherichia coli, le cadre de lecture ouvert MGM101 dépourvue des 22 premiers codons est amplifié par PCR et placé en aval de la séquence malE codant pour la protéine de liaison du maltose (MBP), dans une version modifiée du vecteur d&#39;expression pMAL-C2E. Ceci génère la fusion MBP-Mgm101 avec un élément de liaison contenant un site de clivage pour la protéase de PreScission (figure 3). Le plasmide est construit d&#39;abord en sélectionnant E. transformants coli sans IPTG et la sélection bleu / blanc Xgal. Le plasmide pMAL-C2E-MGM101 résultant est ensuite introduit dans le E. coli BL21-CodonPlus (DE3)-RIL en sélectionnant l&#39;ampicilline et les colonies résistantes au chloramphénicol. 1. Expression, Induction, lyse cellulaire et DNase I Traitement <ol><li> Inoculer transformants fraîches dans 10 ml de milieu LB (1% Bactotryptone, 0,5% d&#39;extrait de levure, NaCl 1%) additionné de glucose (0,2%), l&#39;ampicilline (100 pg / ml) et le chloramphénicol (50 pg / ml). Incuber à 37 ° CO / N avec agitation à 200 rpm. </li><li> Inoculer le 10 ml de préculture dans 2 litres de milieu LB supplémenté comme ci-dessus. Cultiver les cellules à 37 ° C avec agitation jusqu&#39;à DO600 atteigne 0,5. </li><li> Induire l&#39;expression de la protéine de fusion MBP-Mgm101 en ajoutant isopropyl β-D-1-thiogalactopyranoside (IPTG) à une concentration finale de 0,3 mM. Cultiver les cellules avec pendant 5 heures sous agitation à 30 ° C. </li><li> Recueillir les cellules par centrifugation à l&#39;aide d&#39;un Beckman JA-10 rotor (5500 xg, 4 ° C, 10 min). Après élimination du surnageant, remettre en suspension le culot cellulaire dans 30 ml de tampon de lyse (20 mM Tris-HCl, pH 7,4 et 1 mM EDTA, pH 7,4) contenant des inhibiteurs de la protéase (25 pM de leupeptine, 1 pM de pepstatine A et 1 mM PMSF). </li><li> Soniquer suspension cellulaire sur la glace pendant 20sec à l&#39;aide d&#39;un processeur ultra-sons (Systèmes de chaleur; modèle W-385) au cycle de service de 50%, laisser refroidir sur glace pendant 30 secondes, et répétez 2x. </li><li> Ajouter 1 ml de DNAse 1 stock à 2 mg / ml. Basculer le lysat de cellules à 4 ° C pendant 2 heures. </li><li> Ajuster NaCl à une concentration finale de 500 mM. </li><li> Retirer les débris cellulaires par centrifugation à 10 000 xg à 4 ° C pendant 30 min. </li></ol> 2. Purification avec amylose chromatographie d&#39;affinité <ol><li> Equilibrer 1,5 ml de résine d&#39;amylose (50% suspension) dans le tampon de lyse. Ajouter la résine d&#39;amylose équilibrée au lysat cellulaire. Balancer doucement à 4 ° CO / N. </li><li> Chargez le mélange lysat résine sur une colonne de chromatographie Econo-Colonne installé dans une chambre froide. Laisser les protéines non liées au passage de la colonne par gravité. </li><li> Laver la résine amylose avec 300 ml de tampon de lavage I (20 mM Tris-HCl, pH 7,4, NaCl 400 mM, EDTA 1 mM, pH 7,4, et 0,2 mM PMSF). </li><li> Répétez le lavage avec 150 ml de tampon de lavage II (20 mM TRis-HCl, pH 7,4, NaCl 200 mM, EDTA 1 mM, pH 7,4, et 0,2 mM PMSF). </li><li> Ajouter 5 ml de tampon d&#39;élution (20 mM Tris-HCl, pH 7,4, NaCl 200 mM, EDTA 1 mM, pH 7,4, et 0,2 mM PMSF, 10 mM maltose) à la colonne, incuber à 4 ° C pendant 10 min, recueillir l&#39;éluat et répéter pour plus de fois. </li><li> Combinez les éluats, déterminer le rendement et la pureté de MBP-Mgm101 en chargeant une aliquote de l&#39;éluat sur ​​un gel SDS-PAGE suivie d&#39;une coloration de Coomassie (Figure 4). </li></ol> 3. PreScission clivage de la protéase et Chromatographie d&#39;échange de cations <ol><li> Ajouter 50 unités de protéase PreScission à l&#39;éluat MBP-Mgm101. Effectuer le clivage à 4 ° CO / N et lui permettre de continuer pendant la dialyse contre le tampon de dialyse (20 mM Tris-HCl, pH 7,4, NaCl 100 mM, DTT 2 mM, EDTA 1 mM, pH 7,4, et 0,2 mM PMSF) </li><li> Contrôler l&#39;efficacité de clivage sur un gel SDS-PAGE (figure 5A). </li><li> Chargez le clivage MBP-Mgm101 par multiple injecterions sur une cartouche S Mini Macro-Prep Haute Bio-Scale connectés à un système FPLC DuoFlow biologique Bio-Rad. </li><li> Lancer la chromatographie d&#39;échange de cations en appliquant un gradient par étape en sel de 5 mM, 300 mM, 500 mM, 750 mM et 1,000 mM de NaCl qui est créé par le mélange tampon A (NaCl 5 mM, 10 mM de phosphate de Na, pH 7,2; 1 mM PMSF) et le tampon B (1 M NaCl, 10 mM phosphate de sodium, pH 7,2, 1 mM PMSF). Régler le taux de débit de 0,5 ml / min. Recueillir la fraction Mgm101 et contrôler la pureté de la protéine sur un gel SDS-PAGE (figure 5B). </li></ol> 4. Chromatographie d&#39;exclusion stérique <ol><li> Réunir les fractions protéiques de la chromatographie d&#39;échange de cations. Dialyser la protéine dans le tampon d&#39;équilibrage GF (MOPS 20 mM, pH 7,0, NaCl 150 mM, EDTA 1 mM, pH 7,4, 5 mM de 2-mercaptoéthanol, 0,2 mM PMSF). </li><li> Concentrer la protéine avec colonne de centrifugation ultrafiltration 15R VIVASPIN de réduire le volume de ~ 1 ml par centrifugation à 3000 xg à 4 ° C. </li><li> Chargez Mgm101 ona calibré Superose 6 prep colonne de qualité équilibrée avec le tampon de chromatographie (20 mM MOPS, pH 7,0, NaCl 150 mM, 5 mM β-mercaptoéthanol, 1 mM EDTA, pH 7,4, 0,2 mM PMSF). </li><li> Lancer l&#39;exclusion de taille avec le tampon de chromatographie fonctionner à un débit de 0,5 ml / min. </li><li> Recueillir les fractions des pics Mgm101 purifiés. Charger des aliquotes des fractions sur un 12% SDS-PAGE pour vérifier la qualité finale de la protéine. </li><li> Concentré de la protéine avec VIVASPIN 6, puis congeler rapidement les échantillons dans N2 liquide et conserver à -80 ° C. </li><li> Utilisez les Mgm101 échantillons dans les 6-12 mois pour le dosage de fixation d&#39;ADNss (Figure 8) et pour la visualisation de structure par microscopie électronique à transmission (figure 9). </li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+properly+configured+ring+structure+is+critical+for+the+function+of+the+mitochondrial+DNA+recombination+protein.">A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein.</a> Mgm101. J. Biol. Chem. 287, 37259-37268 (2012).</li><li>Zuo, X., Xue, D., Li, N., Clark-Walker, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+core+of+the+mitochondrial+genome+maintenance+protein+Mgm101p+in+Saccharomyces+cerevisiae+determined+with+a+temperature-conditional+allele.">A functional core of the mitochondrial genome maintenance protein Mgm101p in Saccharomyces cerevisiae determined with a temperature-conditional allele.</a> FEMS Yeast Res. 7, 131-140 (2007).</li><li>Itoh, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+packaging+proteins+Glom+and+Glom2+coordinately+organize+the+mitochondrial+nucleoid+of+Physarum+polycephalum.">DNA packaging proteins Glom and Glom2 coordinately organize the mitochondrial nucleoid of Physarum polycephalum.</a> Mitochondrion. 11, 575-586 (2011).</li><li>Janicka, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+RAD52-like+single-stranded+DNA+binding+protein+affects+mitochondrial+DNA+repair+by+recombination.">A RAD52-like single-stranded DNA binding protein affects mitochondrial DNA repair by recombination.</a> Plant J. 72, 423-435 (2012).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Il a été un défi pour produire une protéine stable, native recombinante Mgm101 de E. coli peut-être en raison de son insolubilité dans les cellules bactériennes. Dans ce rapport, nous montrons que la stratégie MBP-fusion permet à la protéine d&#39;être exprimée à un niveau raisonnablement élevé. En utilisant une coloration microscopie électronique à transmission négative et la chromatographie d&#39;exclusion stérique, nous avons précédemment montré que la protéine MBP-fusion forme des oli...

La recombinaison homologue est important pour la réparation des cassures double-brin (CDB) et interbrins réticule, et pour la reprise de la réplication de l&#39;ADN à partir des fourches de réplication effondrées 1. En recombinaison homologue conventionnelle, la réaction central est catalysée par les recombinases ATP-dépendants dont RecA chez les procaryotes et Rad51 et Dmc1 chez les eucaryotes 1-3. Ces recombinases former des filaments nucléoprotéiques sur ssDNA, qui sont essentiels pour initier recherche d&#39;homologie et l&#39;invasion de brin au sein de matrices d&#39;ADN duplex (figure 1, cadre de gauche) 4-7. En plus du schéma classique, la recombinaison homologue peut également avoir lieu d&#39;une manière RecA/Rad51-independent (figure 1, cadre de droite). Par exemple, la levure RAD52 et Rad59 protéines peuvent directement catalyser le recuit de brins d&#39;ADN simple brin complémentaires qui sont exposées par la résection des pauses ADN double brin. Ce processus de recombinaison, connu sous le nom chanterLe fil recuit, généralement n&#39;implique pas appariement homologue des modèles ADN double brin. Après recuit, les queues hétérologues sont éliminés par des exonucléases et pseudos sont ligués pour restaurer 8-10 continuité du génome. Réparation par le mécanisme de recuit simple brin est souvent accompagnée de suppressions de séquences génomiques entre les régions directement répétées. Rad52 appartient à un groupe diversifié de protéines de recombinaison qui sont répandues parmi les bactériophages 11. Ces protéines sont également connus comme simples protéines recuit Strand (SSAP), en fonction de leur activité dans la promotion de l&#39;hybridation de molécules d&#39;ADN simple brin homologue simples. Les meilleurs SSAP de bactériophages sont caractérisés Redβ et Erf du bactériophages λ et P22, RecT du prophage rac et la protéine Sak du phage lactococcal UL36. Les SSAP sont structurellement caractérisées par un pli β-β-β-α typique, bien similitude est pratiquement indétectablepouvoir dans leurs séquences primaires. Tous ces pays sont de grands anneaux homo-oligomères de 10-14 symétrie in vitro 12-14. Les conséquences fonctionnelles de cet ordre organisation structurelle plus caractéristique n&#39;est pas bien comprise. Nous sommes intéressés à comprendre le mécanisme de recombinaison homologue dans le génome mitochondrial. Nous avons déjà identifié le gène MGM101 qui est essentielle pour le maintien de l&#39;ADNmt dans Saccharomyces cerevisiae 15. MGM101 a ensuite été trouvée associée avec nucléoïdes mitochondriales et est nécessaire pour la tolérance de l&#39;ADNmt à des agents endommageant l&#39;ADN 16. Cependant, l&#39;étude de Mgm101 a été retenu dans la dernière décennie par la difficulté de produire Mgm101 recombinant. Nous avons récemment réussi à produire Mgm101 soluble à de grandes quantités de E. coli en utilisant la stratégie de fusion MBP. Cela nous a permis de démontrer que Mgm101 actionssimilitudes biochimiques et structurales avec la Rad52-famille de protéines 17,18. Dans ce rapport, une procédure de purification en trois étapes est décrite, qui produit Mgm101for analyses biochimiques et structurales homogènes (figure 2).

<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Expression vector pMAL-c2E</td>
			<td>New England Biolabs</td>
			<td>#N8066S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PreScission Protease</td>
			<td>GE Healthcare Life Sciences</td>
			<td>#27-0843-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BL21-CodonPlus(DE3)-RIL cells</td>
			<td>Strategene</td>
			<td>#230245</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Leupeptin</td>
			<td>Roche Applied Science</td>
			<td>#11034626001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Roche Applied Science</td>
			<td>#11359053001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Roche Applied Science</td>
			<td>#10837091001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Sigma</td>
			<td>#DN25-1G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Isopropyl &beta;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Roche Applied Science</td>
			<td>#11411446001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England Biolabs</td>
			<td>#E8021L</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Econo-Column chromatography column</td>
			<td>BIO-RAD</td>
			<td>#7372512</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bio-Scale Mini Macro-Prep High S cartridge (1 ml)</td>
			<td>BIO-RAD</td>
			<td>#732-4130</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 15R Ultrafiltration spin column (10,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS15RH02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Superose 6 prep grade column</td>
			<td>Amersham Bioscirnces</td>
			<td>#17-0489-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VIVASPIN 6 Ultrafiltration spin column (5,000 MWCO)</td>
			<td>Sartorius Stedium</td>
			<td>#VS0611</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

preparation of the mgm101 recombination protein by mbp-based tagging strategy

Le gène a été identifié MGM101 il ya 20 ans pour son rôle dans le maintien de l&#39;ADN mitochondrial. Études réalisées dans plusieurs groupes ont suggéré que la protéine Mgm101 est impliqué dans la réparation par recombinaison de l&#39;ADN mitochondrial. Des études récentes ont indiqué que Mgm101 est lié à la famille des protéines de recombinaison Rad52 type. Ces protéines forment des anneaux oligomères et de promouvoir l&#39;hybridation de molécules d&#39;ADN simple brin homologue simples. Cependant, la caractérisation des Mgm101 a été entravée par la difficulté à produire la protéine recombinante. Ici, une procédure fiable pour la préparation recombinante de Mgm101 est décrite. Protéine liant le maltose (MBP) étiquetée Mgm101 est d&#39;abord exprimé dans Escherichia coli. La protéine de fusion est d&#39;abord purifié par chromatographie d&#39;affinité en amylose. Après avoir été libéré par clivage protéolytique, Mgm101 est séparée de MBP par chromatographie d&#39;échange cationique. Monodispersé Mgm101 est alors obtenuepar chromatographie d&#39;exclusion de taille. Un rendement de ~ 0,87 mg de Mgm101 par litre de culture bactérienne peut être obtenue régulièrement. Le Mgm101 recombinante a un minimum de contamination de l&#39;ADN. Les échantillons préparés sont utilisés avec succès pour biochimiques, structurelles et unique analyse d&#39;images de particules de Mgm101. Ce protocole peut aussi être utilisé pour la préparation d&#39;autres grandes protéines liant l&#39;ADN oligomères qui peuvent être mal repliées et toxique pour les cellules bactériennes.

Mgm101 est une protéine de recombinaison Rad52 liée dans les mitochondries. RAD52 a été largement étudié pour son rôle dans la recombinaison de l&#39;ADN mitochondrial (Figure 1). Recombinant Mgm101 peut être préparé par une procédure en trois étapes (figure 2). Ceci est facilité par l&#39;utilisation de la stratégie MBP-tagging qui permet Mgm101 pour être exprimé sous une forme soluble et ensuite libéré de l&#39;étiquette par clivage protéolytique (figure 3)...

Watch this Scientific Journal Video about Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy at JoVE.com

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

La protéine de nucléoïde mitochondrial de la levure, Mgm101, est une protéine de recombinaison Rad52 type qui forme des gros anneaux oligomères. Un protocole est décrit pour préparer Mgm101 recombinant soluble avec la protéine de liaison du maltose (MBP)-tagging stratégie couplée avec échange de cations et la chromatographie d&#39;exclusion stérique.

Préparation de la recombinaison de protéines Mgm101 par la stratégie de marquage MBP basée

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

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Biology

12.0K vues.  State University of New York Upstate Medical University. La protéine de nucléoïde mitochondrial de la levure, Mgm101, est une protéine de recombinaison Rad52 type qui forme des gros anneaux oligomères. Un protocole est décrit pour préparer Mgm101 recombinant soluble avec la protéine de liaison du maltose (MBP)-tagging stratégie couplée avec échange de cations et la chromatographie d'exclusion stérique.

Vidéo: Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

Coloration des protéines dans les gels de Coomassie G-250, sans solvant et l&#39;acide acétique organique

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Recherche

Biologie

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utiliser le système de fractionnement GELFREE 8100 pour les Poids Moléculaire à base de fractionnement avec récupération de la phase liquide

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Identifier les effets des mutations du gène BRCA1 sur la recombinaison homologue en utilisant les cellules qui expriment endogène de type sauvage du gène BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Modification génétique et la recombinaison des cultures d&#39;organes salivaires Gland

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering homologues constructions de recombinaison<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Améliorer le taux de réussite de cristallisation de protéines par hasard Microseed Matrice dépistage

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein base Expression dépistage des protéines membranaires dans<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and &#947;-radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4&#039;,6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans

This protocol demonstrates how to track a protein nuclear translocation under heat stress by using a green fluorescence protein (GFP) fusion protein as a marker and 4',6-diamidino-2-phenylindole (DAPI) staining. The DAPI staining protocol is fast and preserves the GFP and protein subcellular localization signals.

Détection de protéines localisation subcellulaire par Green Fluorescence marquage de protéines et 4', 6-Diamidino-2-phénylindole coloration chez Caenorhabditis elegans

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Détection et caractérisation de l'auto-assemblage des protéines in Vivo par la cytométrie des flux

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Un test de chimiotaxie amélioré pour l’identification rapide des chimioattractifs rhizobactériens dans les exsudats racinaires

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...