Ce travail a été financé par Human Frontier Science Program de subvention RGP0024.

1. Préparation Patron de l&#39;ADN et la transcription in vitro <ol><li> Le gène d&#39;intérêt est cloné dans T7 et / ou, par exemple SP6 promoteur transcriptionnel. </li><li> Pour la transcription in vitro de la matrice d&#39;ADN est linéarisé par une enzyme de restriction appropriée de coupe en aval du codon stop ORF et / ou de l&#39;ARNm extrémité 3 &#39;. Il faut vérifier linéarisation complète de l&#39;ADN plasmidique en exécutant le produit de digestion de restriction à l&#39;électrophorèse sur gel d&#39;agarose. </li><li> Le plasmide linéarisé est utilisé pour en réaction de transcription in vitro. Concentration différente de la matrice ADN peuvent être testés pour déterminer la concentration d&#39;ADN optimale requise pour la transcription in vitro. En règle générale, avec Ambion Trousse MEGAscript à haut rendement de transcription (Ambion / Life Technologies, Grand Island, NY), 1 pg d&#39;ADN linéarisé donne 40-60 ug d&#39;ARNm. La transcription in vitro se fait suivant les instructions du fabricant (Ambion / Life Technologies, Grand Island, État de New York). </li><li> Après transcription in vitro de l&#39;ARNm est purifié par précipitation au chlorure de lithium selon les instructions du fabricant (Ambion de MEGAscript Kit Haut de transcription de rendement). </li><li> l&#39;intégrité ARNm est encore vérifiée par électrophorèse sur des gels d&#39;acrylamide ou d&#39;agarose. </li></ol> 2. Traduction cellulaire in vitro gratuit Pour la traduction in vitro en utilisant R abbit R eticulocyte Lysate, RRL (Promega, Madison, WI), suivez les étapes ci-dessous: <ol><li> Préparer 100 pi dans l&#39;enseignement de traduction in vitro la réaction constructeur suivante. En bref, dans la nucléase l&#39;eau libre, ajouter 2 pl mélange d&#39;acides aminés moins méthionine (1 mM), 10 unités d&#39;inhibiteur de RNase (Invitrogen / Life Technologies, Grand Island, NY), 20 pCi de matières radioactives [35 S]-méthionine (MP Biomedicals, Solon, OH), 70 pi de lysat de réticulocytes de lapin (Promega, Madison, WI). </li><li> Incuber eréaction e à 30 ° C pendant 5 min à l&#39;avance au chaud la réaction de traduction. Ajouter 2 ug d&#39;ARNm de la réaction de traduction et incuber à 30 ° C pendant 5 min. </li><li> Arrêter la réaction en plaçant la réaction de traduction sur la glace. </li></ol> 3. Isolement de polypeptides naissants de réaction de traduction in vitro <ol><li> À isoler polypeptide naissant lié aux ribosomes (polysomes), réaction de traduction est couché au-dessus de 4,5 ml de glycérol 30% dans 10 mM de Tris-HCl de pH 7,6 tampon, contenant 100 mM, KCl 10 mM MgCI 2, et centrifugé à 100 000 xg dans un TLA-110 du rotor (Beckman Coulter, Inc, Brea, CA) pendant 1 h à 4 ° C. </li><li> Le surnageant est encore soigneusement enlevées. </li><li> Le culot contenant polysomique polypeptides naissants est remis en suspension dans un petit volume (10-15 pi) de 1 mM pH tampon Tris-HCl 7,6, contenant 0,5 mg / ml ribonucléase A, (Invitrogen / Life Technologies, Grand Island, NY) et incubées pendant 30 min à 37 ° C. Dans l&#39;ordrepour améliorer l&#39;hydrolyse de la liaison ester d&#39;peptidyl-ARNt, NaOH est ajouté à une concentration finale de 10 mM, et l&#39;incubation est poursuivie pendant 30 minutes supplémentaires. </li></ol> Réaction de traduction in vitro assemblés sans ARNm et réalisée dans les mêmes conditions (suivie de l&#39;isolement de peptides naissants comme cela a été décrit) servirait de contrôle majeur. Le traitement de la réaction de traduction (s) à la puromycine avant de soumettre l&#39;extrait à une centrifugation en gradient de densité peut servir comme un contrôle supplémentaire. 4. Résoudre le polypeptide naissant sur Tris-tricine SDS-PAGE <ol><li> Les polypeptides isolés naissantes sont résolus et analysés sur Tris-tricine SDS-PAGE. S&#39;il vous plaît noter que le montant de la matière chargée sur le gel dépend de l&#39;efficacité de l&#39;étiquetage des protéines, l&#39;efficacité sur la traduction des protéines et de nombreux autres paramètres. La quantité de matière chargée doit être ajustée de façon empirique pour permettre aux besvisualisation t et la séparation des différents naissantes chaînes polypeptidiques. </li><li> Après les gels d&#39;électrophorèse sont fixés, séché à l&#39;aide de gel sous vide teinturier et soumis à une autoradiographie. La distribution des polypeptides naissants sont observées à l&#39;aide phosphoimaging. </li></ol> 5. Les résultats représentatifs Bovine Gamma-B cristallin a été traduit en lysat de réticulocytes de lapin ainsi que dans E. coli S30 Systèmes Extrait (Promega, Madison, WI), suivie par l&#39;isolement des chaînes polypeptidiques naissantes. La figure 1 illustre les étapes impliquées dans l&#39;isolement des ribosomes liés chaînes naissantes. Dans la présente étude, le but était de tester, en cas de changement de l&#39;environnement du répertoire c.-à-traduction de ARNt (connu pour être sensiblement différente chez les mammifères (RRL) et procaryotes (E. coli) des systèmes en raison de différences dans les biais de codon entre les organismes) peut modifier le mouvement du ribosome le long d&#39;ARNm et de la distribution des sites de pause de traduction.Altered mouvement ribosome devrait conduire à des changements dans la distribution des tailles des polypeptides naissants qui s&#39;accumulent pendant la traduction, ainsi que leurs intensités relatives. Les intensités relatives des bandes de refléter la durée de la pause. Les polypeptides naissants ont été isolés et résolus sur les 16,5% T, 6% C Tris-tricine SDS-PAGE (Figure 2). Gamma-B cristallin est une protéine de 20 kDa. L&#39;observation des polypeptides naissants de non-identiques longueurs et des intensités de RRL et E. coli systèmes indique que le mouvement le long des ribosomes ARNm dans ces deux systèmes suivants cinétique de traduction différents. Par exemple, nous avons observé une bande importante de 17,7 kDa dans E. lysat coli et non dans le système de RRL, ce qui suggère qu&#39;il existe un site de pause additionnel de translation dans la région 3&#39;-fin de l&#39;ARNm (encodage région C-terminale de Gamma B cristallin), quand il est traduit en E. coli système. Nous notons que Gamma-B ports cristallin tandem Arg codons (AGG 153 et AGA 154) qui sont fréquentes dans les systèmes mammaliens, mais sont connus pour être extrêmement rare et lentement traduit dans E. coli. un nouveau peptide naissant s&#39;accumulent dans E. système coli (~ 17,7 kDa Figure 2.2) est probablement causée par le ribosome en s&#39;arrêtant à ces codons rares en tandem. S&#39;il vous plaît noter que pas de bandes peuvent être observés en l&#39;absence de l&#39;ARNm (Figure 2.3) et que le traitement puromycine supprime la plupart des chaînes naissantes de polysomes (Figure 2.3). Un traitement prolongé à la puromycine (&gt; 15 min) supprime toutes les chaînes naissantes (données non présentées). Cela confirme que les produits observés polypeptidiques sont associés des chaînes naissantes ribosome, plutôt que mRNP cosedimenting aux polysomes. Comme mentionné ci-dessus, le biais de codon est l&#39;organisme ainsi que des tissus spécifiques. L&#39;exemple représentatif décrit ici, indique clairement que le changement de l&#39;environnement du répertoire c.-à-traduction de ARNt / codonbiais peut conduire à des mouvements modifié du ribosome le long de l&#39;ARNm. Évidemment, cette technique simple peut non seulement permettre l&#39;identification des sites de pause de translation le long d&#39;ARNm, mais permet également une comparaison rapide des sites de pause de traduction entre les différents systèmes. <img alt="Figure 1" src="/files/ftp_upload/4026/4026fig1.jpg" /> Figure 1. Schéma expliquant les étapes à suivre pour isoler les ribosomes liés polypeptides naissants. Bovine Gamma-B cristallin séquence ORF (528 paires de bases) a été cloné en aval du promoteur T7 dans pBSKS +. Pour la transcription in vitro de la matrice d&#39;ADN est linéarisé en le traitant avec EcoRI. Modèle linéarisé a été utilisé pour la transcription in vitro. Promoteur T7 dans la matrice d&#39;ADN est reconnue par l&#39;ARN polymérase T7 qui transcrit l&#39;aval Gamma-gène B cristallin. ARNm est purifié en utilisant la méthode de précipitation au chlorure de lithium. L&#39;ARNm purifié est utilisé pour la traduction in vitro. Suivantd&#39;incubation, la réaction est la traduction en couches sur le dessus de la solution de glycérol à 30% et centrifugé pendant 1 h à 4 ° C à 100.000 X g d&#39;isoler ribosome liés polypeptides naissants. <img alt="Figure 2" src="/files/ftp_upload/4026/4026fig2.jpg" /> Figure 2. Isolement des bovins polypeptides cristallines Gamma-B naissantes traduites en lysat de réticulocytes de lapin (RRL) et E. Lysate coli. 2 mRNA pg a été mélangé dans 100 ul de réaction contenant RRL ou E. coli S30 Extrait System (Promega, Madison, WI) selon les instructions du fabricant avec 20 pCi de [35 S]-méthionine et incubées à 30 ° C pendant 5 min. Après l&#39;incubation de la réaction de traduction a été superposé au-dessus de 4,5 ml de glycérol 30% dans 10 mM de pH tampon Tris-HCl 7,6, contenant 100 mM, KCl 10 mM MgCI 2, et centrifugé pendant 1 h à 4 ° C et 100.000 X g dans une TLA-rotor 110 (Beckman Coulter, Inc, Brea, CA). Le culot a été isolé polyribosome resuspended dans 20 pi de 1 mM de Tris-HCl pH 7,6, contenant 0,5 mg / ml ribonucléase A (Invitrogen / Life Technologies, Grand Island, NY), suivie par un traitement avec NaOH (comme décrit dans la section du protocole). Polypeptides naissants ont été résolus sur les 16,5% T, 6% C Tris-Tricine SDS-PAGE sur gel. Le gel a été séché et exposé pour autoradiographie. Suite à l&#39;exposition du gel a été numérisé à l&#39;aide du scanner de GE Healthcare Imaging Typhoon. La figure 2.1 montre le gel avec des polypeptides naissants isolés et résolus après des réactions de traduction fait dans deux systèmes différents (RRL et E. coli). Figure 2.2 Le gel a été analysée par l&#39;image Quant TL ., logiciels v2005 et utilisé ici comme un exemple pour montrer que le poids moléculaire et l&#39;intensité des polypeptides naissants qui s&#39;accumulent dans deux systèmes peuvent être facilement analysé et comparé la figure 2.3 montre deux ensembles de contrôles: les polypeptides naissants isolés et résolus sur SDS-PAGE après la traduction réactions ont été assembléssans ARNm et / ou après les réactions de conversion ont été traitées avec la puromycine (5 min, concentration finale. 1 mM) avant la réaction a été soumis à une centrifugation.

<ol>
	<li>Komar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pause+for+thought+along+the+co-translational+folding+pathway.">A pause for thought along the co-translational folding pathway.</a> Trends Biochem. Sci. 34, 16-24 (2009).</li><li>Sharp, P. M., Cowe, E., Higgins, D. G., Shields, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Codon+usage+patterns+in+Escherichia+coli,+Bacillus+subtilis,+Saccharomyces+cerevisiae,+Schizosaccharomyces+pombe,+Drosophila+melanogaster+and+Homo+sapiens;+a+review+of+the+considerable+within-species+diversity.">Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity.</a> Nucleic Acids Res. 16, 8207-8210 (1988).</li><li>Dittmar, K. A., Goodenbour, J. M., Pan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+differences+in+human+transfer+RNA+expression.">Tissue-specific differences in human transfer RNA expression.</a> PLoS Genet. 2, e221 (2006).</li><li>Ikemura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Codon+usage+and+tRNA+content+in+unicellular+and+multicellular+organisms.">Codon usage and tRNA content in unicellular and multicellular organisms.</a> Mol. Biol. Evol. 2, 13-34 (1985).</li><li>Wolin, S. 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Pas de conflits d&#39;intérêt déclarés.

Pour obtenir des résultats reproductibles, la qualité et la concentration des composants utilisés pour la transcription in vitro et les réactions de traduction sont essentielles. Dans l&#39;étude actuelle, nous avons utilisé des kits disponibles dans le commerce et les extraits qui fournissent des données hautement reproductibles, s&#39;il est manipulé avec soin. Cependant, la traduction-compétentes extraits peuvent être préparés à partir de la cellule de son choix, si nécessaire. Qualit?...

<table border="1"><tbody><tr><td> Nom de réactif / Kit </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> MEGAscript T7 haute Kit de transcription rendement </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Inhibiteur de la ribonucléase </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Label </td><td> MP Biomedicals </td><td> 0151006 </td></tr><tr><td> Ribonucléase-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> De réticulocytes de lapin Lysate système, nucléase traité </td><td> Promega </td><td> L4960 </td></tr><tr><td> E. coli S30 Extrait système pour les modèles linéaires </td><td> Promega </td><td> L1030 </td></tr><tr><td> Centrifugation </td><td> Beckman Coulter </td><td> Optima TLX Ultracentrifugeuse </td></tr><tr><td> Luminophore de mémorisation autoradiographiey </td><td> GE Healthcare </td><td> Typhoon 9410 imageur mode variable </td></tr><tr><td> Logiciel pour l&#39;analyse du polypeptide naissant </td><td> GE Healthcare </td><td> Quant image TL, v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

Le taux d&#39;allongement de translation n&#39;est pas uniforme. ARNm structure secondaire, l&#39;usage des codons et ARNm des protéines associées peuvent modifier le mouvement du ribosome sur le message pour revue, voir 1. Cependant, il est maintenant largement admis que l&#39;usage des codons synonymes est la principale cause de non-uniformes des taux d&#39;allongement de translation 1. Codons synonymes ne sont pas utilisés avec une fréquence identique. Un biais existe dans l&#39;utilisation des codons synonymes avec certains codons utilisés plus fréquemment que d&#39;autres 2. Codon biais est l&#39;organisme ainsi que le tissu spécifique 2,3. En outre, la fréquence de l&#39;usage des codons est directement proportionnelle à la concentration des ARNt apparenté 4. Ainsi, un codon fréquemment utilisé aura plus multitude de ARNt correspondants, ce qui implique en outre que d&#39;un codon fréquentes seront traduits plus rapidement qu&#39;une rares. Ainsi, les régions sur l&#39;ARNm enrichi en codons rares (sites de pause potentiels) seront, en règle ralentir le mouvement du ribosome sur le message d&#39;accumulation et de la cause des peptides naissants des tailles respectives 5-8. Ces sites de pause peut avoir un impact fonctionnel sur l&#39;expression des protéines, stabilité de l&#39;ARNm et le repliement des protéines pour revue, voir 9. En effet, il a été montré que l&#39;atténuation de ces sites de pause peut altérer le mouvement du ribosome sur l&#39;ARNm et par la suite peut affecter l&#39;efficacité du co-traductionnelle repliement des protéines (in vivo) 1,7,10,11. Pour comprendre le processus de repliement des protéines in vivo, dans la cellule, qui est finalement couplé au processus de synthèse des protéines, il est essentiel d&#39;obtenir un aperçu complet sur ​​l&#39;impact du codon d&#39;utilisation / ARNt contenu sur le mouvement des ribosomes le long de l&#39;ARNm en cours d&#39;élongation translationnelle . Nous décrivons ici une technique simple qui peut être utilisé pour localiser les principaux sites de pause de traduction pour un ARNm donné traduit en diverses des systèmes acellulaires 6-8. Cette procédure est basée sur l&#39;isolement des polypeptides naissante accumulating sur les ribosomes lors de la traduction in vitro d&#39;un ARNm cible. La logique est celle à basse fréquence des codons, l&#39;augmentation du temps de séjour des résultats ribosomes dans des quantités accrues de peptides naissants des tailles correspondantes. Dans ARNm transcrit in vitro est utilisé pour des réactions de translation in vitro de la présence d&#39;acides aminés marqués radioactivement pour permettre la détection des chaînes naissantes. Afin d&#39;isoler les ribosomes liés naissantes complexes polypeptidiques la réaction de traduction est posée sur le dessus de la solution de glycérol à 30% suivie d&#39;une centrifugation. Polypeptides naissants dans polysomique granulés sont traités avec la ribonucléase A et résolus par SDS-PAGE. Cette technique peut être éventuellement utilisées pour une protéine et permet l&#39;analyse de mouvement le long du ribosome ARNm et la détection des sites de pause principaux. En outre, ce protocole peut être adapté pour étudier les facteurs et conditions qui peuvent altérer le mouvement du ribosome et donc potentiellement peut également modifier ee fonction / conformation de la protéine.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Une technique pour identifier les sites de pause translationnelle sur l&#39;ARNm est décrite. Cette procédure est basée sur l&#39;isolement des polypeptides naissants qui s&#39;accumulent sur les ribosomes lors de la traduction in vitro d&#39;un ARNm cible, suivie par l&#39;analyse de la taille des chaînes naissantes en utilisant une électrophorèse sur gel dénaturant.

Isolement du ribosome Bound polypeptides naissants<em&gt; In vitro</em&gt; Pour identifier les sites de pause de translation le long d&#39;ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

isolation-ribosome-bound-nascent-polypeptides-vitro-to-identify

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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

16.2K vues.  Cleveland State University. Une technique pour identifier les sites de pause translationnelle sur l'ARNm est décrite. Cette procédure est basée sur l'isolement des polypeptides naissants qui s'accumulent sur les ribosomes lors de la traduction in vitro d'un ARNm cible, suivie par l'analyse de la taille des chaînes naissantes en utilisant une électrophorèse sur gel dénaturant.

Vidéo: Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - Une méthode pour identifier transcriptome à l&#39;échelle des sites de liaison des protéines liant l&#39;ARN

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

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Biologie

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

L&#39;isolement et la In vitro Activation des Caenorhabditis elegans Sperme

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Aide de la portée d&#39;identifier le potentiel Motifs de la réglementation dans les gènes co-régulés

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcription in vitro et plafonnement de l&#39;ARNm de luciférase Gaussia Suivie par la transfection de cellules HeLa

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome Bound

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

La synthèse in vitro de l&#39;ARNm modifié pour l&#39;induction de l&#39;expression des protéines dans les cellules humaines

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Mesurer le stress oxydatif Résistance<em&gt; Caenorhabditis elegans</em&gt; En 96 plaques de microtitration bien

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Examen des protéines liées à Nascent ADN dans les cellules de mammifères utilisant la technique BrdU-Chip-Slot-Ouest

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Un protocole pour l’utilisation de gènes série d’analyses d’enrichissement pour identifier le modèle Animal approprié de recherche translationnelle

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

In Vitro Dosage de SUMOylation pour étudier l’activité de Ligase E3 SUMO

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...