We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the &#8220;priority programmes&#8221; SPP1316 and SPP1617.

<ol>
	<li>Simpson, C. G., Brown, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+extension+assay.">Primer extension assay.</a> Methods Mol. Biol. 49, 249-256 (1995).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 (1977).</li><li>Fekete, R. A., Miller, M. J., Chattoraj, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+labeled+oligonucleotide+extension:+a+rapid+and+quantitative+protocol+for+primer+extension.">Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension.</a> Biotechniques. 35, 90-94 (2003).</li><li>Schuster, C. F., 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=Characterization+of+a+mazEF+Toxin-Antitoxin+Homologue+from+Staphylococcus+equorum.">Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum.</a> J. Bacteriol. 195, 115-125 (2013).</li><li>Nolle, N., Schuster, C. F., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+paralogous+yefM-yoeB+loci+from+Staphylococcus+equorum+encode+functional+toxin-antitoxin+systems.">Two paralogous yefM-yoeB loci from Staphylococcus equorum encode functional toxin-antitoxin systems.</a> Microbiology. 159, 1575-1585 (2013).</li><li>Yamaguchi, Y., Park, J. H., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+in+bacteria+and+archaea.">Toxin-antitoxin systems in bacteria and archaea.</a> Annu. Rev. Genet. 45, 61-79 (2011).</li><li>Schuster, C. F., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+are+ubiquitous+and+versatile+modulators+of+prokaryotic+cell+fate.">Toxin-antitoxin systems are ubiquitous and versatile modulators of prokaryotic cell fate.</a> FEMS Microbiol. Lett. 340, 73-85 (2013).</li><li>Goeders, N., Van Melderen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+as+multilevel+interaction+systems.">Toxin-antitoxin systems as multilevel interaction systems.</a> Toxins. 6, 304-324 (2014).</li><li>Yamaguchi, Y., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+interferases,+sequence-specific+endoribonucleases+from+the+toxin-antitoxin+systems.">mRNA interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems.</a> Progress in molecular biology and translational science. 85, 467-500 (2009).</li><li>Park, J. H., Yamaguchi, Y., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+MazF-bs+(EndoA)+is+a+UACAU-specific+mRNA+interferase.">Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase.</a> FEBS Lett. 585, 2526-2532 (2011).</li><li>Zhu, L., 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=et+al.Staphylococcus+aureus+MazF+specifically+cleaves+a+pentad+sequence,+UACAU,+which+is+unusually+abundant+in+the+mRNA+for+pathogenic+adhesive+factor+SraP.">et al.Staphylococcus aureus MazF specifically cleaves a pentad sequence, UACAU, which is unusually abundant in the mRNA for pathogenic adhesive factor SraP.</a> J. Bacteriol. 191, 3248-3255 (2009).</li><li>Zhu, L., 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=The+mRNA+interferases,+MazF-mt3+and+MazF-mt7+from+Mycobacterium+tuberculosis+target+unique+pentad+sequences+in+single-stranded+RNA.">The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA.</a> Mol. Microbiol. 69, 559-569 (2008).</li><li>Fu, Z., Donegan, N. P., Memmi, G., Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+MazFSa,+an+endoribonuclease+from+Staphylococcus+aureus.">Characterization of MazFSa, an endoribonuclease from Staphylococcus aureus.</a> J. Bacteriol. 189, 8871-8879 (2007).</li><li>Marmur, 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+procedure+for+the+isolation+of+deoxyribonucleic+acid+from+micro-organisms.">A procedure for the isolation of deoxyribonucleic acid from micro-organisms.</a> J. Mol. Biol. 3, 208-218 (1961).</li><li>Emory, S. A., Belasco, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ompA+5'+untranslated+RNA+segment+functions+in+Escherichia+coli+as+a+growth-rate-regulated+mRNA+stabilizer+whose+activity+is+unrelated+to+translational+efficiency.">The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency.</a> J. Bacteriol. 172, 4472-4481 (1990).</li><li>Cole, S. T., Bremer, E., Hindennach, I., Henning, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+promoters+for+the+ompA+gene+which+encodes+a+major+outer+membrane+protein+of+Escherichia+coli.">Characterisation of the promoters for the ompA gene which encodes a major outer membrane protein of Escherichia coli.</a> Mol. Gen. Genet. 188, 472-479 (1982).</li><li>Schleifer, K. H., Kilpper-B&#228;lz, R., Devriese, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+arlettae+sp.+nov.,+S.+equorum+sp.+nov.+and+S.+kloosii+sp.+nov.:+Three+New+Coagulase-Negative,+Novobiocin-Resistant+Species+from+Animals.">Staphylococcus arlettae sp. nov., S. equorum sp. nov. and S. kloosii sp. nov.: Three New Coagulase-Negative, Novobiocin-Resistant Species from Animals.</a> Syst. Appl. Microbiol. 5, 501-509 .</li><li>Yu, H., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HIV-1+and+avian+myeloblastosis+virus+reverse+transcriptase+fidelity+on+RNA+and+DNA+templates.">Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates.</a> J. Biol. Chem. 267, 10888-10896 (1992).</li><li>Ying, B. W., Fourmy, D., Yoshizawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+the+use+of+radioactivity+by+fluorescence+for+biochemical+studies+of+RNA.">Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA.</a> RNA. 13, 2042-2050 (2007).</li></ol>

The authors have nothing to disclose that would present a conflict of interest.

Fluoreszierende Primerverlängerung ist ein einfaches und schnelles Verfahren zur Bestimmung der 5&#39;-Enden der RNAs, entweder für TSP- oder sekundäre RNA-Prozessierung Identifikation. Durch die Verwendung von fluoreszierenden Primern können die Umsetzungen eingerichtet werden und ausgeführt werden, ohne zusätzliche Sicherheitseinrichtungen (anders als im Fall von radioaktiv markierten Primern). Da die Proben durch Fluoreszenz detektiert, können sie abgebildet werden, während der Elektrophorese durchgeführt wi...

Primerverlängerungs 1 ist ein Molekular Methode, um die 5&#39;-Enden der spezifischen RNA-Moleküle bis zu einer eine Basisauflösung bestimmen. Der Vorteil gegenüber anderen Verfahren, wie 5&#39;-RACE (schnelle Amplifikation von cDNA-Enden) ist die schnelle Durchlaufzeit und die Fähigkeit, leicht zu analysieren, die eine Mischung von verschiedenen Längen von RNA-Molekülen. Diese Methode funktioniert, indem RNA-Moleküle, die Transkription Reaktionen Reverse mit spezifischen fluoreszierenden Primer, Erzeugung cDNA-Fragmente bestimmter Längen. Diese cDNA-Moleküle werden neben der traditionellen Sanger-Sequenzierungsreaktionen 2 auf denaturierenden Polyacrylamid-Gelen und durch ihre Fluoreszenz aufgrund der Verwendung von fluoreszenzmarkierten Primern nachgewiesen werden. Die Längen der cDNA-Fragmente werden dann durch Vergleich mit der Sequenzierungsleiter beurteilt, so dass die Zuordnung der 5&#39;-RNA-Ende. Traditionell werden Primer-Verlängerungsreaktionen in Verbindung verwendet werdenmit radioaktiven Isotopen, um cDNA-Moleküle auf Röntgenfilmen zu erkennen. Wegen der Gesundheitsrisiken, Entsorgungsprobleme und der einfachen Handhabung, neuere Protokolle zu nutzen Fluoreszenz zum Nachweis der Primer-Extension mit automatisierten Sequenzer, wenn ihre Empfindlichkeit ist etwas niedriger. Mit fluoreszenzmarkierten Primern kann die wiederkehrende Radiomarkierungsverfahren verzichtet werden, als fluoreszierende Primer sind für eine lange Zeit (mehr als ein Jahr in unseren Händen) stabil. Die Methode, die wir hier beschreiben, nutzt ein automatisiertes Gel Sequenzer, aber mit leichten Modifikationen können Kapillare Sequenzern auch für die cDNA Trennung und Detektion 3 verwendet werden. Die Parallelität der Gel-Analyse ermöglicht es, auch eine kleine Menge der RNA-Spaltung oder Verarbeitung zu erfassen. Ein weiterer Vorteil ist die hohe Auflösung dieses Verfahrens als terminale Spaltung oder Verarbeitung von nur einer Basis detektiert werden. In Bezug auf den Nachweis von RNA-Spaltung oder Verarbeitung, typically zwei verschiedene Arten von Primer-Extensions unterschieden. In einem Fall wird die enzymatische Behandlung in vitro durchgeführt unter Verwendung gereinigter RNA und gereinigtes Enzym, wohingegen in dem anderen Fall, wird die Verarbeitung in vivo durchgeführt und die resultierende RNA wird gereinigt. In beiden Fällen wird die RNA unterzogen, um eine Primer-Extension durchgeführt in vitro, jedoch in Abhängigkeit von der Quelle der RNA wird das Verfahren entweder bezeichnet eine in vitro oder in vivo Primerverlängerung. In dem Protokoll wir hier konzentrieren wir uns lediglich auf der in-vivo-Primerverlängerung, aufgrund der Einfachheit der Nutzung (keine gereinigten Proteine ​​notwendig) und die Möglichkeit zur Transkriptionsstartpunkte und die Verarbeitung in der gleichen Zeit zu bestimmen. Allerdings sind in vitro Primerverlängerungen im Prinzip die gleiche Weise eingestellt und dieses Protokoll kann als Ausgangspunkt dienen. Das hier dargestellte Verfahren kann auf viele Bakterienarten, solange sie zugänglich sind hoch angewendetReinheit und High-Yield-Vorbereitung von Nukleinsäuren. Die Forschung in unserem Labor konzentriert sich auf den Regelungsbereich von Toxin-Antitoxin (TA) Systeme 4,5, einem Bereich, in dem die Primer-Extension-Methode wird intensiv genutzt. TA-Systeme sind kleiner in prokaryotischen Genom, das aus einem stabilen und endogen aktive toxische Protein und einer meist instabil Protein oder RNA-Antitoxin, die Toxizität entgegen 6,7 bestehen vorliegenden genetischen Elemente. Toxinaktivität wird manchmal durch Hemmung der Replikation, der Zellwandsynthese oder andere Mechanismen, die von RNase-Aktivität 8,9 ausgeübt wird, aber meistens. Typischerweise wird RNase Spezifität durch die Durchführung verschiedener Tests, von denen der eine der Primer-Extension-Methode bestimmt. Primer-Verlängerungsreaktionen sind für diese Anwendung gut geeignet, da eine Mischung aus gespaltenen und in voller Länge Fragmente können gleichzeitig analysiert werden, um festzustellen ihren 5&#39;-Enden. Verwendung einer Mischung von in vitro und in vivo Primerverlängerungen, diespezifische Toxin RNase-Spaltung, zB Sequenzspezifität bestimmt 10-13 werden. <img alt="Figur 1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> Abbildung 1. Übersicht der Primerverlängerungsverfahren. Bakterielle Kulturen werden nach den experimentellen Anforderungen inkubiert und behandelt. Gesamt-RNA wird aus den Zellen mit DNase I behandelt, um DNA-Spuren zu entfernen, und einer Umkehrtranskriptionsreaktion unter Verwendung von Ziel-DNA spezifischen Fluoreszenz Primer wodurch cDNA unterworfen extrahiert. Genomische DNA oder Plasmide extrahiert und anschließend zur Fluoreszenz Sanger-Sequenzierungsreaktionen zum Größenvergleich mit den cDNA-Fragmenten verwendet. Primerverlängerungsprodukte sind neben Sanger-Sequenzierungsprodukte auf einem denaturierenden Harnstoff-Polyacrylamidgel laufen und mit einem automatisierten Laser und Mikroskop analysiert. Die Sequenzierung Basis, die Linie mit dem cDNA-Band ist die last Basis des 5 &#39;cDNA Ende (blauer Pfeil). Weitere Informationen im Fekete et al. 3 <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</a> Eine Übersicht über den gesamten Primerverlängerungsverfahren kann in 1 gefunden werden. Kurz gesagt, kultiviert, geerntet sind bakterielle Zellen, das Zellpellet lysiert und die RNA extrahiert. Gereinigte RNA wird dann mit DNase I behandelt, um Spuren von DNA-Molekülen, die als Matrizen für die reverse Transkriptase wirken könnten, zu entfernen. Spezifische fluoreszierende Primer an die RNA gegeben, um die Region von Interesse hybridisiert und anschließend revers transkribiert, wodurch einzelsträngige komplementäre DNA (cDNA). Ein Sequenzierleiter wird von traditionellen Sanger-Sequenzierung erstellt die Verwendung von Fluoreszenz Primer und auf einem denaturierenden Polyacrylamid-Gel neben der Primerverlängerung cDNA-Fragmente getrennt. Das resultierendeGel wird durch den Vergleich der Fluoreszenzbanden, die eine Identifizierung des 5&#39;-Enden von Interesse analysiert. Transkriptionsstartpunkte und Verarbeitungsstellen werden dann einzeln durch Sequenzvergleiche geprüft.

<table border="0" cellpadding="0" cellspacing="0" width="1200">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;"><a name="RANGE!A1:D16">Name</a></td>
			<td style="width:196px;">Supplier</td>
			<td style="width:244px;">Catalog Number(s)</td>
			<td style="width:363px;">Comment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">AMV Reverse Transcriptase (20-25 U/&#181;l)</td>
			<td style="width:196px;">NEB / Roche</td>
			<td style="width:244px;">NEB: M0277-T / Roche: 10109118001</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:399px;">DNase I (RNase free)</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2222</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">FastPrep-24 Instrument&#160;</td>
			<td style="width:196px;">MPBio</td>
			<td align="right">116004500</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:399px;">Fluorescently labeled primers</td>
			<td style="width:196px;">Biomers</td>
			<td style="width:244px;">n/a</td>
			<td style="width:363px;">5&#8217; DY-681 modification of &#8220;ordinary&#8221; DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Li-Cor 4200 Sequencer incl. ImagIR Data collection software</td>
			<td style="width:196px;">Li-Cor</td>
			<td style="width:244px;"></td>
			<td style="width:363px;">Product discontinued</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">NanoDrop 2000</td>
			<td style="width:196px;">Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Nuclease free water</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM9915G</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Plasmid mini preparation kit</td>
			<td style="width:196px;">QIAGEN</td>
			<td align="right">12125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RapidGel-XL-40% Concentrate</td>
			<td style="width:196px;">USB</td>
			<td style="width:244px;">US75863</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">RNA STORAGE BUFFER</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td>AM7000</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Roti-Aqua-P/C/I</td>
			<td style="width:196px;">Carl Roth</td>
			<td style="width:244px;">X985.3</td>
			<td style="width:363px;">Alternative: &#8220;Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)&#8221; from Ambion (AM9720)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">SUPERase&#8226;In RNase Inhibitor</td>
			<td style="width:196px;">Ambion (life technologies)</td>
			<td style="width:244px;">AM2696</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:399px;">Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP</td>
			<td style="width:196px;">GE Healthcare</td>
			<td style="width:244px;">RPN2538</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">TRIzol reagent</td>
			<td style="width:196px;">life technologies</td>
			<td style="width:244px;">15596-026</td>
			<td style="width:363px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:399px;">Zirconia/Silica Beads 0.1 mm</td>
			<td style="width:196px;">BioSpec</td>
			<td style="width:244px;">11079101z</td>
			<td style="width:363px;"></td>
		</tr>
	</tbody>
</table>

fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo

Fluoreszenzbasierten Primer-Extension (FPE) ist ein Molekular Methode, um Transkriptionsstartpunkte oder Verarbeitungsstätten von RNA-Molekülen zu bestimmen. Dies wird durch die reverse Transkription der RNA von Interesse mit spezifischen fluoreszenzmarkierten Primern und anschließender Analyse der resultierenden cDNA-Fragmente durch denaturierende Polyacrylamid-Gelelektrophorese erreicht. Gleichzeitig wird eine traditionelle Sanger-Sequenzierungsreaktion auf dem Gel laufen gelassen, um die Enden der cDNA-Fragmente anzeigen, um deren genaue entsprechenden Basen. Im Gegensatz zu 5&#39;-RACE (Rapid Amplification von cDNA-Enden), wobei das Produkt muss kloniert und sequenziert mehreren Kandidaten kann die Masse der cDNA-Fragmente durch Primerverlängerung erzeugt werden gleichzeitig in einem Gel laufen detektiert werden. Zusätzlich kann das gesamte Verfahren (von der reversen Transkription der endgültigen Analyse der Ergebnisse) an einem Arbeitstag abgeschlossen sein. Durch die Verwendung fluoreszierend markierter Primer, die Verwendung von gefährlichen radioaktiven Isotop markierten Reagenzienvermieden werden und die Bearbeitungszeit verringert werden als Produkte können während der Durchführung der Elektrophorese nachgewiesen werden. In dem folgenden Protokoll beschreiben wir ein in vivo Fluoreszenz Primer-Extension-Methode auf das 5&#39;-Ende der RNA schnell und zuverlässig zu erfassen, abzuleiten Transkriptionsstartpunkte und RNA-Verarbeitungsstellen (zB durch Toxin-Antitoxin-Systemkomponenten) in S. aureus, E. coli und anderen Bakterien.

Wie in 6 dargestellt, kann ein Primer-Verlängerungsreaktion verwendet werden, um die Transkriptionsstartpunkte von Transkripten von Interesse zu bestimmen, und kann helfen, Promotorregionen ableiten (typischerweise -10 bis -35 Elemente identifiziert). Die oberste (längste) cDNA Fragment stellt das 5&#39;-Ende der mRNA und somit leicht zugeordnet werden, wenn im Vergleich zu der Sequenzierungsleiter werden. <img alt="Figur 6" src="/files/ftp_upload/52134/52134fig6highres.jpg...

Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Fluoreszenzbasierten Primer Extension-Technik, um Transkriptionsstartpunkte und Schnittstellen von RNasen Bestimmen<em&gt; In Vivo</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

fluorescence-based-primer-extension-technique-to-determine

Research

JoVE Journal

Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

26.8K Aufrufe.  University of Tübingen. We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

Serie: Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Proboscis Extension Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth (labellum), there are hair-like structures called sensilla which houses taste neurons. When an attractive substance makes contact to the labellum, the fly extends its proboscis to consume the material. Proboscis Extension Response (PER) assay measures this taste behavior response, and it is a useful method to learn about food preferences in a single fly. Solutions of various sugars, such as sucrose, glucose and fructose, are very attractive to the fly. The effect of aversive substances can also be tested as reduction of PER when mixed in a sweet solution.Despite the simplicity of the basic procedure, there are many things that can prevent it from working. One of the factors that requires attention is the fly's responsive state. The required starvation time to bring the fly to the proper responsive state varies drastically from 36 to 72 hours. We established a series of controls to evaluate the fly's state and which allows screening out of non-responsive or hyper-responsive individual animals. Another important factor is the impact level and the position of the contact to the labellum, which would be difficult to describe by words. This video presentation demonstrates all these together with several other improvements that would increase the reproducibility of this method.

Proboscis Extension Response PER Assay in Drosophila

Proboscis extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the proboscis makes contact with an attractive substance, the fly extends its proboscis to consume the substance. Solutions of various sugars are very attractive to the fly.

Proboscis Erweiterung Response (PER) Assay in Drosophila

Proboscis extension response (PER) is a taste behavior assay that has been used in flies as well as in honeybees.
On the surface of the fly's mouth ...

Forschung

Biologie

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopic Fluoreszenz-Tomographie für In-vivo Imaging der Entwicklungsländer Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Primer Extension Capture: Targeted Sequence Retrieval from Heavily Degraded DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ancient bones. The method greatly reduces sample destruction and sequencing demands relative to direct PCR or shotgun sequencing approaches. We used this method to reconstruct the complete mitochondrial DNA (mtDNA) genomes of five Neandertals from across their geographic range. The mtDNA genetic diversity of the late Neandertals was approximately three times lower than that of contemporary modern humans. Together with analyses of mtDNA protein evolution, these data suggest that the long-term effective population size of Neandertals was smaller than that of modern humans and extant great apes.

We present a method of targeted ancient DNA sequence retrieval, which we used to reconstruct the complete mitochondrial genomes of five Neandertal individuals. Comparison of these sequences with present day humans suggests that Neandertals had a long term low effective population size.

Primer Extension Capture: Gezielte Sequence Retrieval von stark degradierte DNA Sources

We present a method of targeted DNA sequence retrieval from DNA sources which are heavily degraded and contaminated with microbial DNA, as is typical of ...

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

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

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

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

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 ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

Einzel-Molekül-Imaging der Genregulation<em&gt; In vivo</em&gt; Verwenden cotranslationalen Aktivierung durch Spaltung (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

Eine quantitative Assay zur Protein Studie: DNA-Wechselwirkungen, Discover Transkriptionsregulatoren der Genexpression, und identifizieren Novel Anti-Tumor-Wirkstoffe

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Ein Proteoliposom Basierte Efflux-Assay auf Einzelmoleküleigenschaften von Cl Bestimmen<sup&gt; -</sup&gt; Kanäle und Transporter

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

Methoden Alternative Promoter Verwendung und Transkriptions-Verordnung von Murine Bcrp1 zu entdecken

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

In Vitro und In Vivo Ansätze Darm Epithelzelle Durchlässigkeit zu bestimmen

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Profilierung der H3K4me3-Modifikation in Pflanzen mittels Spaltung unter Targets und Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...