Diese Arbeit wurde vom Human Frontier Science Program RGP0024 Zuschuss finanziert.

1. DNA-Schablone Herstellung und in vitro Transkription <ol><li> Das Gen von Interesse unter T7 und / oder zB SP6-Transkriptions-Promotor kloniert. </li><li> Für in-vitro-Transkription der Matrizen-DNA mit einem geeigneten Restriktionsenzym-Schnittstellen stromabwärts des Stop-Codon ORF und / oder mRNA 3&#39;-Ende linearisiert. Man braucht, um eine vollständige Linearisierung der Plasmid-DNA durch Ausführen des Restriktionsverdau Produkt auf Agarose-Gelelektrophorese überprüft. </li><li> Das linearisierte Plasmid wird für die in vitro-Transkriptions-Reaktion verwendet. Verschiedene Konzentrationen der Ziel-DNA getestet, um eine optimale DNA-Konzentration für die in vitro Transkription erforderlich zu identifizieren. Im Allgemeinen mit MEGAscript Hohe Ambions Ertrag Transcription Kit (Ambion / Life Technologies, Grand Island, NY), ergibt 1 pg DNA linearisiert 40-60 pg mRNA. In-vitro-Transkription wird nach Herstellerangaben (Ambion / Life Technologies, Grand Islan getand, NY). </li><li> Nach in vitro Transkription wird die mRNA durch Lithiumchlorid Niederschläge nach Herstellerangaben (Ambion die MEGAscript High Yield Transcription Kit) gereinigt. </li><li> mRNA-Integrität wird weiterhin durch Elektrophorese auf Acrylamid oder Agarosegelen überprüft. </li></ol> 2. In vitro Zell Freie Übersetzung Für in vitro Translation mit R abbit R eticulocyte Lysat, RRL (Promega, Madison, WI) die folgenden Schritte aus: <ol><li> Es werden 100 ul in vitro Translation Reaktion nach Herstellerangaben. Kurz gesagt, in Nuclease freies Wasser hinzufügen, 2 ul Aminosäurenmischung minus Methionin (1 mM), 10 Einheiten RNase-Inhibitor (Invitrogen / Life Technologies, Grand Island, NY), 20 uCi von radioaktivem [35 S]-Methionin (MP Biomedicals, Solon, OH), 70 ul Kaninchen-Retikulozytenlysat (Promega, Madison, WI). </li><li> Inkubieren the Reaktion bei 30 ° C für 5 min auf die warme Reaktion Übersetzung voreingestellt. In 2 ug der mRNA an das Translations-Reaktion und Inkubieren bei 30 ° C für 5 min. </li><li> Stoppen Sie die Reaktion, indem Sie die Übersetzung Reaktion auf Eis. </li></ol> 3. Isolierung der entstehenden Polypeptide in vitro Translationsreaktion <ol><li> Um entstehenden Polypeptid gebunden an Ribosomen (Polysomen) zu isolieren, wird die Übersetzung Reaktion auf von 4,5 ml 30% Glycerin in 10 mM Tris-HCl-Puffer pH 7,6 geschichtet, enthaltend 100 mM KCl, 10 mM MgCl 2 und zentrifugiert bei 100.000 × g in einem TLA-110-Rotor (Beckman Coulter, Inc., Brea, CA) für 1 Stunde bei 4 ° C </li><li> Der Überstand wird weiter vorsichtig entfernt. </li><li> Die Polysomen Pellet, das entstehende Polypeptide ist in einem kleinen Volumen (10-15 ul) von 1 mM Tris-HCl-Puffer pH 7,6, resuspendiert, das 0,5 mg / ml Ribonuklease A (Invitrogen / Life Technologies, Grand Island, NY), und inkubiert 30 min bei 37 ° C Umdie Hydrolyse der Peptidyl-tRNA-Ester-Bindung zu erhöhen, wird NaOH auf eine Endkonzentration von 10 mM zugegeben und die Inkubation für weitere 30 min fortgesetzt. </li></ol> In-vitro-Translation Reaktion zusammengebaut ohne mRNA und durchgeführt unter den gleichen Bedingungen (gefolgt von der Isolierung der entstehenden Peptide wie es beschrieben wurde) würde als große Kontrolle dienen. Die Behandlung der Übersetzung Reaktion (en) mit Puromycin vor den Extrakt einer Dichtegradientenzentrifugation kann als zusätzliche Kontrolle zu dienen. 4. Die Lösung der entstehenden Polypeptid auf Tris-Tricin SDS-PAGE <ol><li> Die isolierten naszierenden Polypeptiden werden aufgelöst und analysiert auf Tris-Tricin SDS-PAGE. Bitte beachten Sie, dass die Menge des Materials auf dem Gel wird abhängig von der Effizienz der Markierung von Proteinen, Protein-Effizienz auf Übersetzung des viele andere Parameter geladen. Die Menge des geladenen Materials sollte empirisch eingestellt werden, um die BES erlaubent-Visualisierung und Trennung einzelner naszierenden Polypeptidketten. </li><li> Nach der Elektrophorese Gele fixiert sind, trocknet mit Vakuum-Gel Färber und einer Autoradiographie unterzogen. Die Verteilung der entstehenden Polypeptide werden beobachtet, wenn Phosphoimaging. </li></ol> 5. Repräsentative Ergebnisse Rinder-gamma-B Crystallin wurde in Kaninchen-Retikulozytenlysat übersetzt und in E. coli S30-Extrakt Systems (Promega, Madison, WI), gefolgt von der Isolierung des naszierenden Polypeptidketten. Abbildung 1 zeigt die Schritte bei der Isolierung von Ribosomen gebundenen naszierenden Ketten beteiligt. In der vorliegenden Studie war das Ziel zu überprüfen, ob die Änderung der Umgebung der Übersetzung bedeutet Repertoire von tRNAs (bekanntlich wesentlich verschiedenen in Säugetierzellen (RRL) und prokaryotischen (E. coli)-Systeme aufgrund von Unterschieden in Codon Vorspannung zwischen der Organismen) kann verändern Ribosom Bewegung entlang mRNA bewirken und die Verteilung der Übersetzung Pause Sites.Altered Ribosom Bewegung sollte auf Veränderungen in der Verteilung der Größen der entstehenden Polypeptide akkumulieren während der Übersetzung sowie deren relative Intensitäten führen. Relative Intensitäten der Banden spiegeln die Dauer der Pause. Die entstehenden Polypeptide wurden isoliert und gelöst auf 16,5% T, 6% C Tris-Tricin SDS-PAGE (Abbildung 2). Gamma-B Crystallin ist ein 20 kDa Protein. Die Beobachtung des entstehenden Polypeptide von nicht identischen Längen und Intensitäten in RRL und E. coli-Systemen zeigt, dass die Bewegung entlang der Ribosomen-mRNA in diesen zwei Systeme unterschiedliche Übersetzung Kinetik folgt. Zum Beispiel beobachten wir eine prominente Bande von 17,7 kDa in E. coli-Lysat und nicht in RRL-System, was darauf hindeutet, dass es eine zusätzliche translatorische Pause-Stelle im 3&#39;-Ende der mRNA Bereich (Codierung C-terminale Region von Gamma B Crystallin), wenn es in E. übersetzt coli-System. Wir weisen darauf hin, dass Gamma-B Crystallin Häfen Tandem-Arg-Codons (AGG 153 und AGA 154), die häufigsten sind in Säugetier-Systeme, sondern sind dafür bekannt, extrem selten und langsam übersetzt in E. coli. Ein Roman entstehenden Peptid, der sich in E. coli-System (~ 17,7 kDa Abbildung 2.2) wird wahrscheinlich durch Ribosomen-Pause bei diesen seltenen Codons Tandem verursacht. Bitte beachten Sie, dass keine Bänder in der Abwesenheit von mRNA (Abbildung 2.3) beobachtet werden kann und dass Puromycin Behandlung löscht die meisten der naszierenden Ketten von Polysomen (Abb. 2.3). Eine längere Behandlung mit Puromycin (&gt; 15 min) entfernt alle naszierenden Ketten (Daten nicht gezeigt). Dies bestätigt, dass die beobachteten Polypeptidprodukte Ribosom assoziierten naszierenden Ketten, anstatt mRNPs cosedimenting mit Polysomen sind. Wie oben erwähnt, ist Codon Bias Organismus als auch gewebespezifisch. Der Vertreter hier beschriebenen Beispiel zeigt deutlich, dass die Änderung der Umgebung der Übersetzung heißt Repertoire von tRNAs / CodonBias kann zu veränderten Bewegung des Ribosoms an der mRNA entlang führen. Offensichtlich kann diese einfache Technik ermöglichen nicht nur die Identifizierung der translationalen Pause Standorten entlang mRNA, sondern erlaubt auch eine schnelle Vergleich der Übersetzung Pause Standorten zwischen verschiedenen Systemen. <img alt="1" src="/files/ftp_upload/4026/4026fig1.jpg" /> Abbildung 1. Schematische Erläuterung der Schritte zu isolieren Ribosom gebundenen naszierenden Polypeptide beteiligt sind. Rinder-gamma-B Crystallin ORF-Sequenz (528 Basenpaare) wurde stromabwärts des T7-Promotors kloniert in pBSKS +. Für in-vitro-Transkription der Matrizen-DNA wurde durch Behandlung mit EcoRI linearisiert. Linearisierte Template wurde für in vitro-Transkription verwendet. T7-Promotor in der DNA-Vorlage wird durch T7-RNA-Polymerase, die die stromabwärtigen Gamma-B Crystallin Gen transkribiert erkannt. mRNA wird unter Verwendung Lithiumchlorid Fällungsverfahren. Die gereinigte mRNA ist für in vitro-Translation verwendet. FolgendeInkubation Übersetzung Reaktion auf von 30% Glycerin-Lösung geschichtet und zentrifugiert für 1 Stunde bei 4 ° C bei 100.000 × g für Ribosom gebundenen naszierenden Polypeptide zu isolieren. <img alt="2" src="/files/ftp_upload/4026/4026fig2.jpg" /> Abbildung 2. Isolierung von Rinder-gamma-B Crystallin naszierenden Polypeptiden in Kaninchen-Retikulozyten-Lysat (RRL) und E. übersetzt coli-Lysat. 2 pg mRNA wurde in 100 ul Reaktion mit RRL oder E. gemischt coli S30 Extract System (Promega, Madison, WI) nach Anleitung des Herstellers mit 20 Ci [35 S]-Methionin und bei 30 ° C für 5 min. Nach der Inkubation die Translationsreaktion wurde oben auf 4,5 ml 30% Glycerin in 10 mM Tris-HCl-Puffer pH 7,6 geschichtet, enthaltend 100 mM KCl, 10 mM MgCl 2 und zentrifugiert, für 1 Stunde bei 4 ° C und 100.000 × g in ein TLA-110 Rotor (Beckman Coulter, Inc., Brea, CA). Die isolierte polyribosome Pellet war resuspended in 20 ul von 1 mM Tris-HCl pH 7,6, enthaltend 0,5 mg / ml Ribonuklease A (Invitrogen / Life Technologies, Grand Island, NY) durch Behandlung mit NaOH (wie im Protokoll beschrieben) gefolgt. Nascent Polypeptide wurden auf 16,5% T, 6% C Tris-Tricin SDS-PAGE-Gel aufgetrennt. Das Gel wurde getrocknet und mit für die Autoradiographie. Nach der Belichtung wurde das Gel gescannt mit GE Healthcare Typhoon Imaging Scanner. Abbildung 2.1 zeigt das Gel mit naszierenden Polypeptiden isoliert und gelöst nach der Übersetzung Reaktionen in zwei verschiedenen Systemen (RRL und E. coli) durchgeführt. Abbildung 2.2 Das Gel wurde von Image Quant TL analysiert ., v2005 Software und hier als Beispiel zu zeigen, dass das Molekulargewicht und die Intensität der entstehenden Polypeptide, der sich in beiden Systemen problemlos analysiert werden können und im Vergleich benutzt Abbildung 2.3 zeigt zwei Gruppen von Steuerelementen: naszierenden Polypeptide isoliert und auf SDS-PAGE aufgelöst nach der Übersetzung Reaktionen wurden montiertohne Zugabe von mRNA und / oder nach der Übersetzung Reaktionen wurden mit Puromycin (5 min, Endkonzentration. 1 mM) behandelt, bevor die Umsetzung einer Zentrifugation unterworfen wurde.

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Für reproduzierbare Ergebnisse, Qualität und Konzentration der Komponenten für die in vitro Transkription und Translation-Reaktionen verwendet kritisch sind. In der aktuellen Studie haben wir kommerziell verfügbaren Kits und Extrakte, die in hohem Maße reproduzierbar Daten zu liefern, bei sorgfältiger Behandlung eingesetzt. Jedoch kann die Übersetzung-zuständigen Extrakte aus der Zelle des eigenen Wahl hergestellt werden, wenn nötig. Qualität der mRNA kann die Translation beeinflussen, so ist es von g...

<table border="1"><tbody><tr><td> Name des Reagenz / Kit </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> MEGAscript T7 High Yield Transcription Kit </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Ribonuclease Inhibitor </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> Ribonuclease-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Kaninchen-Retikulozytenlysat-System behandelt, Nuclease </td><td> Promega </td><td> L4960 </td></tr><tr><td> E. coli S30-Extrakt-System für lineare Matrizen </td><td> Promega </td><td> L1030 </td></tr><tr><td> Zentrifugation </td><td> Beckman Coulter </td><td> Optima TLX Ultrazentrifuge </td></tr><tr><td> Speicherleuchtstoffplatte Autoradiogrammy </td><td> GE Healthcare </td><td> Typhoon 9410 Variable Mode Imager </td></tr><tr><td> Software zur Analyse entstehenden Polypeptid </td><td> GE Healthcare </td><td> Bild Quant TL, V2005 </td></tr></tbody></table>

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

Die Geschwindigkeit der Translationselongation ungleichmäßig ist. Sekundärstruktur der mRNA, mRNA-Codon-Nutzung und assoziierten Proteinen kann Ribosom Bewegung auf die Meldung zur Prüfung zu verändern siehe 1. Allerdings ist es nun weitgehend akzeptiert, dass ein Synonym Codonverwendung die primäre Ursache für ungleichmäßige Translationselongation Sätze 1 ist. Synonyme Codons sind nicht mit gleicher Frequenz verwendet. Eine Vorspannung besteht in der Verwendung von synonymen Codons mit Codons einigen häufiger als andere 2 verwendet. Codon-Bias ist Organismus als auch gewebespezifische 2,3. Darüber hinaus ist die Frequenz der Codonverwendung direkt proportional zu den Konzentrationen von verwandten tRNAs 4. Somit wird ein häufigsten verwendeten Codons höhere Vielzahl von entsprechenden tRNAs, die impliziert, dass ein häufiges Codon schneller als einem seltenen wird man übersetzt werden. So werden die Regionen auf mRNA in seltenen Codons (potentiellen Pause Sites) angereichert in der Regel langsamer Bewegung auf dem Ribosom MessaGE und Ursache entstehende Anhäufung von Peptiden der jeweiligen Größen 5-8. Diese Pause Standorte können funktionelle Auswirkungen auf die Protein-Expression haben, finden die mRNA-Stabilität und Faltung von Proteinen für die Überprüfung 9. Tatsächlich wurde gezeigt, dass eine Senkung der solche Pause-Seiten können Ribosom Bewegung auf mRNA verändern und anschließend kann die Effizienz der Co-translationale (in vivo) Proteinfaltung 1,7,10,11 beeinflussen. Um den Prozess der Proteinfaltung in vivo zu verstehen, in der Zelle, die letztlich auf den Prozess der Protein-Synthese ist es wichtig, umfassende Einblicke in die Auswirkungen der Codon-Nutzung / tRNA-Inhalte auf die Bewegung entlang der Ribosomen-mRNA gewinnen während Translationselongation gekoppelt . Hier beschreiben wir eine einfache Technik, die zur großen Pause Übersetzung Standorte für eine bestimmte mRNA in verschiedenen zellfreien Systemen 6-8 übersetzt zu lokalisieren. Dieses Verfahren beruht auf der Trennung entstehenden Polypeptide accumulati Basisng an Ribosomen während der in vitro Translation eines Target-mRNA. Der Grund ist, dass bei niedrigen Frequenzen Codons, die Erhöhung der Verweilzeit der Ribosomen führt zu einer erhöhten Mengen von naszierendem Peptide der entsprechenden Größe. In-vitro-transkribierte mRNA wurde in vitro Translations-Reaktionen in Gegenwart von radioaktiv markierten Aminosäuren die Detektion der naszierenden Ketten zu ermöglichen. Um Ribosom gebundenen Komplexe entstehenden Polypeptid zu isolieren die Übersetzung Reaktion auf von 30% Glycerin-Lösung geschichtet, gefolgt von Zentrifugation. Nascent Polypeptide in polysomaler Pellets werden weiter mit Ribonuklease A behandelt und durch SDS-PAGE. Diese Technik kann für jedes Protein potentiell verwendet werden und ermöglicht die Analyse der Bewegung entlang Ribosom-mRNA und der Nachweis der größten Pause Seiten. Zusätzlich kann dieses Protokoll angepasst, um Faktoren und Bedingungen, die Ribosomen-Bewegung zu verändern und damit potentiell auch verändern th studieren werdene-Funktion / Konformation des Proteins.

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

Eine Technik zur translationalen Pause Sites auf mRNA zu identifizieren beschrieben wird. Dieses Verfahren beruht auf der Trennung entstehenden Polypeptide ansammeln auf Ribosomen während der in vitro Translation einer Ziel-mRNA, durch die Größe Analyse der naszierenden Ketten mit Hilfe eines denaturierenden Gelelektrophorese gefolgt basiert.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

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

Research

JoVE Journal

Biology

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

16.2K Aufrufe.  Cleveland State University. Eine Technik zur translationalen Pause Sites auf mRNA zu identifizieren beschrieben wird. Dieses Verfahren beruht auf der Trennung entstehenden Polypeptide ansammeln auf Ribosomen während der in vitro Translation einer Ziel-mRNA, durch die Größe Analyse der naszierenden Ketten mit Hilfe eines denaturierenden Gelelektrophorese gefolgt basiert.

Serie: 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 - Ein Verfahren zur Transkriptom-weiten die Bindungsstellen des RNA-bindende Proteine ​​Identifizieren

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

Forschung

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.

Isolation und In-vitro- Aktivierung von Caenorhabditis elegans Sperma

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.

Mit SCOPE potenzieller regulatorischer Motive in Coregulated Gene zu identifizieren

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.

In vitro Transkription und Capping von Gaussia-Luciferase mRNA von HeLa-Zelle Transfektion Gefolgt

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.

Mit SECM Arrest Sequenz als Werkzeug zum Ribosom gebunden Polypeptide isolieren

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.

In vitro Synthese von modifizierten mRNA zur Induktion der Proteinexpression in menschlichen Zellen

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.

Mess Oxidativer Stress Widerstandsrat<em&gt; Caenorhabditis elegans</em&gt; In 96-Well-Mikrotiterplatten

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.

Untersuchung von Proteinen, entstehende DNA in Säugerzellen Bound Mit BrdU-chip-Slot-westliche Technik

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.

Ein Protokoll für die Verwendung von Gen Set Anreicherung Analyse zur Ermittlung von geeigneten Tiermodell für die translationale Forschung

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- Sumoylierung Assay SUMO E3 Ligase Tätigkeit zu studieren

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