Die Arbeit wurde von der Universität von Verona im Rahmen des gemeinsamen Projekts 2014 (Charakterisierung der ATL-Genfamilie im Weinstock und sein Engagement im Widerstand gegen Plasmopara Viticola) unterstützt.

<ol>
	<li>Jaillon, O., 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+grapevine+genome+sequence+suggests+ancestral+hexaploidization+in+major+angiosperm+phyla.">The grapevine genome sequence suggests ancestral hexaploidization in major angiosperm phyla.</a> Nature. 449 (7161), 463-467 (2007).</li><li>Adam-Blondon, A. -. 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=.">.</a> Genetics, Genomics, and Breeding of Grapes. , 211-234 (2011).</li><li>Chen, L., Hellmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+E3+Ligases:+Flexible+Enzymes+in+a+Sessile+World.">Plant E3 Ligases: Flexible Enzymes in a Sessile World.</a> Mol. Plant. 6 (5), 1388-1404 (2013).</li><li>Vierstra, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-26S+proteasome+system+at+the+nexus+of+plant+biology.">The ubiquitin-26S proteasome system at the nexus of plant biology.</a> Nat. Rev. Mol. Cell Biol. 10 (6), 385-397 (2009).</li><li>Serrano, M., Parra, S., Alcaraz, L. D., Guzm&#225;n, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ATL+Gene+Family+from+Arabidopsis+thaliana+and+Oryza+sativa+Comprises+a+Large+Number+of+Putative+Ubiquitin+Ligases+of+the+RING-H2+Type.">The ATL Gene Family from Arabidopsis thaliana and Oryza sativa Comprises a Large Number of Putative Ubiquitin Ligases of the RING-H2 Type.</a> J. Mol. Evol. 62 (4), 434-445 (2006).</li><li>Aguilar-Hern&#225;ndez, V., Aguilar-Henonin, L., Guzm&#225;n, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+in+the+Architecture+of+ATLs,+a+Family+of+Plant+Ubiquitin-Ligases,+Leads+to+Recognition+and+Targeting+of+Substrates+in+Different+Cellular+Environments.">Diversity in the Architecture of ATLs, a Family of Plant Ubiquitin-Ligases, Leads to Recognition and Targeting of Substrates in Different Cellular Environments.</a> PLoS One. 6 (8), e23934 (2011).</li><li>Guzm&#225;n, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prolific+ATL+family+of+RING-H2+ubiquitin+ligases.">The prolific ATL family of RING-H2 ubiquitin ligases.</a> Plant Signal Behav. 7 (8), 1014-1021 (2012).</li><li>Grimplet, J., 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+grapevine+gene+nomenclature+system.">The grapevine gene nomenclature system.</a> BMC Genomics. 15, 1077 (2014).</li><li>Prince, V. E., Pickett, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Splitting+pairs:+the+diverging+fates+of+duplicated+genes.">Splitting pairs: the diverging fates of duplicated genes.</a> Nat. Rev. Genet. 3 (11), 827-837 (2002).</li><li>Magadum, S., Nerjee, U., Murugan, P., Gangapur, D., Ravikesavan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+duplication+as+a+major+force+in+evolution.">Gene duplication as a major force in evolution.</a> J. Gen. 92 (1), 155-161 (2013).</li><li>Wang, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+Gene+Duplication+and+Their+Contribution+to+Expansion+of+Gene+Families+in+Grapevine.">Patterns of Gene Duplication and Their Contribution to Expansion of Gene Families in Grapevine.</a> Plant Mol. Biol. Rep. 31 (4), 852-861 (2013).</li><li>Fasoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Grapevine+Expression+Atlas+Reveals+a+Deep+Transcriptome+Shift+Driving+the+Entire+Plant+into+a+Maturation+Program.">The Grapevine Expression Atlas Reveals a Deep Transcriptome Shift Driving the Entire Plant into a Maturation Program.</a> Plant Cell. 24 (9), 3489-3505 (2012).</li><li>. BLAST2.6.0 Available from: <a target="_blank" href="https://blast.ncbi.nlm.nih.gov/Blast.cgi">https://blast.ncbi.nlm.nih.gov/Blast.cgi</a> (2016)</li><li>. Vitis vinifera cv. Corvina gene expression Atlas Available from: <a target="_blank" href="https://www.researchgate.net/publication/273383414_54sample_datamatrix_geneIDs_Fasoli2012">https://www.researchgate.net/publication/273383414_54sample_datamatrix_geneIDs_Fasoli2012</a> (2015)</li><li>. Sequence Read Archive (SRA) Available from: <a target="_blank" href="https://www.ncbi.nlm.nih.gov/sra">https://www.ncbi.nlm.nih.gov/sra</a> (2017)</li><li>Bolger, A. M., Lohse, M., Usadel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trimmomatic:+a+flexible+trimmer+for+Illumina+sequence+data.">Trimmomatic: a flexible trimmer for Illumina sequence data.</a> Bioinformatics. 30 (15), 2114-2120 (2014).</li><li>Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+gapped-read+alignment+with+Bowtie+2.">Fast gapped-read alignment with Bowtie 2.</a> Nat Meth. 9 (4), 357-359 (2012).</li><li>Anders, S., Pyl, P. T., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTSeq-a+Python+framework+to+work+with+high-throughput+sequencing+data.">HTSeq-a Python framework to work with high-throughput sequencing data.</a> Bioinformatics. 31 (2), 166-169 (2015).</li><li>. Version 3.4.1 Available from: <a target="_blank" href="https://www.r-project.org/">https://www.r-project.org/</a> (2017)</li><li>Ritchie, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=limma+powers+differential+expression+analyses+for+RNA-sequencing+and+microarray+studies.">limma powers differential expression analyses for RNA-sequencing and microarray studies.</a> Nucleic Acids Res. 43 (7), e47 (2015).</li><li>Love, M. I., Huber, W., Anders, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderated+estimation+of+fold+change+and+dispersion+for+RNA-seq+data+with+DESeq2.">Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.</a> Genome Biology. 15 (12), 550 (2014).</li><li>Ariani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+characterisation+and+expression+profile+of+the+grapevine+ATL+ubiquitin+ligase+family+reveal+biotic+and+abiotic+stress-responsive+and+development-related+members.">Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members.</a> Sci. Rep. 6, 38260 (2016).</li><li>Vitulo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+deep+survey+of+alternative+splicing+in+grape+reveals+changes+in+the+splicing+machinery+related+to+tissue,+stress+condition+and+genotype.">A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype.</a> BMC Plant Biol. 14 (1), 99 (2014).</li><li>Overbeek, R., Fonstein, M., D'Souza, M., Pusch, G. D., Maltsev, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+gene+clusters+to+infer+functional+coupling.">The use of gene clusters to infer functional coupling.</a> Proc. Natl. Acad. Sci. USA. 96 (6), 2896-2901 (1999).</li><li>Dalquen, D. A., Dessimoz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+Best+Hits+Miss+Many+Orthologs+in+Duplication-Rich+Clades+such+as+Plants+and+Animals.">Bidirectional Best Hits Miss Many Orthologs in Duplication-Rich Clades such as Plants and Animals.</a> Genome Biol. Evol. 5 (10), 1800-1806 (2013).</li><li>Remm, M., Storm, C. E. V., Sonnhammer, E. L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automatic+clustering+of+orthologs+and+in-paralogs+from+pairwise+species+comparisons1.">Automatic clustering of orthologs and in-paralogs from pairwise species comparisons1.</a> J. Mol. Biol. 314 (5), 1041-1052 (2001).</li><li>Kaduk, M., Sonnhammer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+orthology+inference+with+Hieranoid+2.">Improved orthology inference with Hieranoid 2.</a> Bioinformatics. 33 (8), (2017).</li><li>Cramer, G. R., 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=Transcriptomic+analysis+of+the+late+stages+of+grapevine+(Vitis+vinifera+cv.+Cabernet+Sauvignon)+berry+ripening+reveals+significant+induction+of+ethylene+signaling+and+flavor+pathways+in+the+skin.">Transcriptomic analysis of the late stages of grapevine (Vitis vinifera cv. Cabernet Sauvignon) berry ripening reveals significant induction of ethylene signaling and flavor pathways in the skin.</a> BMC Plant Biol. 14, 370 (2014).</li><li>Juretic, N., Hoen, D. R., Huynh, M. L., Harrison, P. M., Bureau, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+fate+of+MULE-mediated+duplications+of+host+gene+fragments+in+rice.">The evolutionary fate of MULE-mediated duplications of host gene fragments in rice.</a> Genome Res. 15 (9), 1292-1297 (2005).</li><li>Filichkin, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mapping+of+alternative+splicing+in+Arabidopsis+thaliana.">Genome-wide mapping of alternative splicing in Arabidopsis thaliana.</a> Genome Res. 20 (1), 45-58 (2010).</li><li>Quesada, V., Macknight, R., Dean, C., Simpson, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoregulation+of+FCA+pre-mRNA+processing+controls+Arabidopsis+flowering+time.">Autoregulation of FCA pre-mRNA processing controls Arabidopsis flowering time.</a> EMBO J. 22 (12), 3142-3152 (2003).</li><li>Wong, D. C. J., Gutierrez, R. L., Gambetta, G. A., Castellarin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+cis-regulatory+element+structure+and+discovery+of+motif-driven+gene+co-expression+networks+in+grapevine.">Genome-wide analysis of cis-regulatory element structure and discovery of motif-driven gene co-expression networks in grapevine.</a> DNA Res. 24 (3), 311-326 (2017).</li><li>Wong, D. C. J., Matus, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+Integrated+Networks+for+Identifying+New+Secondary+Metabolic+Pathway+Regulators+in+Grapevine:+Recent+Applications+and+Future+Opportunities.">Constructing Integrated Networks for Identifying New Secondary Metabolic Pathway Regulators in Grapevine: Recent Applications and Future Opportunities.</a> Front. Plant Sci. 8, 505 (2017).</li></ol>

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In der genomischen Ära wurden viele Genfamilien tief in mehreren Pflanzenarten charakterisiert. Diese Informationen sind Voraussetzung für funktionelle Studien und bieten einen Rahmen, um die Rolle der verschiedenen Mitglieder in einer Familie weiter zu untersuchen. In diesem Zusammenhang ist auch eine Notwendigkeit für eine Nomenklatur-System ermöglicht zur eindeutigen Identifizierung jedes Mitglied in einer Familie, Vermeidung von Redundanz und Verwirrungen, die entstehen können, wenn Namen von verschiedenen Forsc...

In den letzten zehn Jahren wurde viel Forschung in Grapevine Genomics durchgeführt. Weinrebe ist eine anerkannte wirtschaftlich relevanten Ernte, die ein Modell für die Forschung auf Fruchtentwicklung und die Reaktionen von Gehölzen auf biotischen und abiotischen Stress geworden ist. In diesem Zusammenhang die Freilassung von Vitis Vinifera CV PN40024 Genom in 20071 und seine aktualisierte Version in 20112 führte zu einer schnellen Ansammlung von "Omics" angelegte Daten und einen Ausbruch von Hochdurchsatz-Studien. Basierend auf die publizierten Sequenzdaten, die umfassende Analyse einer bestimmten gen-Familie (in der Regel bestehend aus Proteinen Austausch konservierte Motive, strukturelle und funktionelle Gemeinsamkeiten und evolutionären Beziehungen), kann jetzt durchgeführt werden, um zu entdecken sein molekulare Funktionen, Evolution und gen Expressionsprofile. Diese Analysen können tragen zum Verständnis, wie Genfamilien physiologische Prozesse auf eine genomweite Ebene steuern.
Ubiquitin vermittelten Abbau von wichtigen Proteinen, erfordern einen fein abgestimmte Umsatz um regelmäßige zelluläre Prozesse zu gewährleisten, sind viele Aspekte des Lebenszyklus der Anlage geregelt. Wichtige Bestandteile des Ubiquitin-vermittelten Abbauprozess E3 Ubiquitin Ligases, die für die Flexibilität des Systems, durch die Rekrutierung von Einzelzielen3verantwortlich sind. Dementsprechend stellen diese Enzyme eine riesige Genfamilie mit rund 1.400 E3 Ligase-Kodierung Gene in Arabidopsis Thaliana Genom4, jedes E3-Ubiquitin-Ligase handeln für Ubiquitination von zielgruppenspezifischen Proteinen vorhergesagt. Trotz der Bedeutung der Substrat-spezifische Ubiquitination in zellulären Verordnung in Pflanzen ist wenig bekannt über wie Ubiquitination-Signalweg reguliert wird und Zielproteine wurden nur in wenigen Fällen identifiziert. Die Entschlüsselung dieser Spezifität und Verordnung Mechanismen stützt sich zunächst auf die Identifizierung und Charakterisierung der verschiedenen Komponenten des Systems, insbesondere E3 Ligases. Unter Ubiquitin Ligases ist ATL Unterfamilie geprägt von 91 Mitglieder in A. Thaliana anzeigen eine RING-H2 Finger Domäne5,6, einige von ihnen spielt eine Rolle in der Verteidigung und Hormon Antworten7identifiziert.
Der erste entscheidende Schritt zu definieren, die Mitglieder einer neuen gen-Familie ist die genaue Definition der Family-Features, z. B. Konsens Motive Schlüsselbereichen und Protein-Sequenz-Eigenschaften. In der Tat erfordert das zuverlässige Abrufen aller gen Familienmitglieder BLAST Analyse einige obligatorische Sequenz Merkmale in bestimmten Proteins Domänen verantwortlich für Protein-Funktion/Tätigkeit, als Protein-Signatur. Dies kann durch vorherige Charakterisierung derselben Genfamilie in anderen Pflanzenarten erleichtert oder erreicht durch die Analyse verschiedener Gene, die vermeintlich aus der gleichen Familie in verschiedenen Pflanzenarten, allgemeine Sequenzen zu isolieren. Die Familienmitglieder können dann individuell benannt werden, nach gemeinsamen Regeln, die von internationalen Konsortien für einen bestimmten Pflanzenarten besiedelt. Weinrebe zum Beispiel solche Verfahren auf die Empfehlungen des Ausschusses Super-Nomenklatur für Traube gen Annotation (sNCGGa), über den Bau einer phylogenetischen Baum einschließlich V. Vinifera und A. Thaliana unterliegt gen Familienmitglieder gen Anmerkung erlauben ausgehend von Nukleotid-Sequenzen8.
Chromosom Lokalisierung von Familienmitgliedern und gen Doppelarbeit Umfrage ermöglichen das Vorhandensein von Vollständiggenom oder Tandem duplizierten Genen Hervorhebung. Solche Informationen erscheint sinnvoll, vermeintliche Genfunktionen zu lüften, da es vielleicht funktionelle Redundanz zeigen oder unterschiedliche Situationen, d. h., nicht-Funktionalisierung, Neo-Funktionalisierung oder Sub-Funktionalisierung9 zeigen. Beiden Neo - und sub - functionalization sind wichtige Ereignisse, die genetischen Neuheit, Pflanze Anpassung an sich verändernde Umgebungen10neue zelluläre Komponenten zur erstellen. Insbesondere Vervielfältigungen von ancestral Genen und Produktion neuer Gene wurden sehr häufig während der Evolution des Genoms Weinrebe und neugeformten Gene aus proximalen und Tandem Duplikationen in Grapevine waren wahrscheinlicher, produzieren neue Funktionen11.
Ein weiterer wichtiger Faktor bei der Entschlüsselung Familie Genfunktion ist das transkriptomischen-Profil. Die Verfügbarkeit von öffentlichen Datenbanken den Zugriff auf eine riesige Menge an transkriptomischen Daten kann so ausgenutzt werden, um gen Familienmitglieder mit groß angelegten in Silico Expressionsanalysen mutmaßliche Funktionen zuweisen. In der Tat der eigentümliche Ausdruck einiger Gene in bestimmten Pflanzenorganen oder als Reaktion auf bestimmte Belastungen geben einige Hinweise auf die vermeintlichen Rollen der entsprechenden Proteine unter definierten Bedingungen und Hypothesen über mögliche Unterstützung zukommen Sub-Funktionalisierung von duplizierten Genen, verschiedene Herausforderungen zu reagieren. Zu diesem Zweck ist es wichtig, mehrere Datasets zu berücksichtigen: Diese können bereits vorhandene gen Ausdruck Matrizen, wie z. B. die genomweite transkriptomischen Atlas der Weinrebe Organe und Entwicklungsstadien12, oder ad-hoc durch gebaut werden können Abrufen von transkriptomischen Datensätzen für die bestimmten Pflanzenarten definierten Belastungen ausgesetzt. Darüber hinaus können ein einfacher Ansatz mit zwei Matrizen, eines mit paarweisen Ähnlichkeit Daten und eines mit paarweisen Co Ausdruck Koeffizienten angewendet werden um die Beziehungen zwischen Reihenfolge Ähnlichkeit und Ausdruck Muster innerhalb einer Genfamilie zu bewerten.
Diese Arbeit soll einen globalen Ansatz, Definition der Genstruktur, konserviertes Protein Motive, chromosomalen Position, gen Duplikationen und Expressionsmuster, als auch die Vorhersage von Protein Lokalisierung und Phosphorylierung Websites erreichen eine vollständige Charakterisierung einer Genfamilie in Pflanzen. Ein umfassender Ansatz wird hier auf die Charakterisierung der ATL E3 Ubiquitin Ligase Familie in Grapevine angewendet. Entsprechend der neuen Rolle des ATL Unterfamilie Mitglieder bei der Regulierung der wichtige zelluläre Prozesse7, diese Arbeit gut helfen die Identifizierung der starke Kandidaten für funktionelle Studien, und schließlich die molekularen Mechanismen zu entwirren der Anpassung von diesem wichtigen Kulturpflanzen auf seine Umgebung.

<table>
	<tbody>
		<tr>
			<td>Personal computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Local Alignment Search Tool (BLAST)</td>
			<td></td>
			<td></td>
			<td>https://blast.ncbi.nlm.nih.gov/Blast.cgi</td>
		</tr>
		<tr>
			<td>Molecular Evolutionary Genetics Analysis (MEGA)</td>
			<td></td>
			<td></td>
			<td>http://www.megasoftware.net/</td>
		</tr>
		<tr>
			<td>Motif-based sequence analysis tools (MEME)</td>
			<td></td>
			<td></td>
			<td>http://meme-suite.org/</td>
		</tr>
		<tr>
			<td>Geneious</td>
			<td>Biomatters Limited</td>
			<td></td>
			<td>http://www.geneious.com/</td>
		</tr>
		<tr>
			<td>ProtParam Tool</td>
			<td></td>
			<td></td>
			<td>http://web.expasy.org/protparam/</td>
		</tr>
		<tr>
			<td>ngLOC</td>
			<td></td>
			<td></td>
			<td>http://genome.unmc.edu/ngLOC/index.html</td>
		</tr>
		<tr>
			<td>TargetP v1.1 Server</td>
			<td></td>
			<td></td>
			<td>http://www.cbs.dtu.dk/services/TargetP/</td>
		</tr>
		<tr>
			<td>Protein Prowler</td>
			<td></td>
			<td></td>
			<td>http://bioinf.scmb.uq.edu.au:8080/pprowler_webapp_1-2/</td>
		</tr>
		<tr>
			<td>MUsite</td>
			<td></td>
			<td></td>
			<td>http://musite.sourceforge.net/</td>
		</tr>
		<tr>
			<td>Pfam</td>
			<td></td>
			<td></td>
			<td>http://pfam.xfam.org/</td>
		</tr>
		<tr>
			<td>TMHMM Server v. 2.0</td>
			<td></td>
			<td></td>
			<td>http://www.cbs.dtu.dk/services/TMHMM/</td>
		</tr>
		<tr>
			<td>ProtScale</td>
			<td></td>
			<td></td>
			<td>http://web.expasy.org/protscale/</td>
		</tr>
		<tr>
			<td>Grape Genome Database (CRIBI)</td>
			<td></td>
			<td></td>
			<td>http://genomes.cribi.unipd.it/grape/</td>
		</tr>
		<tr>
			<td>PhenoGram</td>
			<td></td>
			<td></td>
			<td>http://visualization.ritchielab.psu.edu/phenograms/plot</td>
		</tr>
		<tr>
			<td>MCScanX</td>
			<td></td>
			<td></td>
			<td>http://chibba.pgml.uga.edu/mcscan2/</td>
		</tr>
		<tr>
			<td>Interactive Tree Of Life (iTOL)</td>
			<td></td>
			<td></td>
			<td>http://itol.embl.de/</td>
		</tr>
		<tr>
			<td>UniProt</td>
			<td></td>
			<td></td>
			<td>http://www.uniprot.org/</td>
		</tr>
		<tr>
			<td>Phylogeny.fr</td>
			<td></td>
			<td></td>
			<td>http://www.phylogeny.fr/index.cgi</td>
		</tr>
		<tr>
			<td>MUSCLE</td>
			<td></td>
			<td></td>
			<td>http://www.ebi.ac.uk/Tools/msa/muscle/</td>
		</tr>
		<tr>
			<td>Gblocks Server</td>
			<td></td>
			<td></td>
			<td>http://molevol.cmima.csic.es/castresana/Gblocks_server.html</td>
		</tr>
		<tr>
			<td>Vitis vinifera cv. Corvina gene expression Atlas datamatrix</td>
			<td></td>
			<td></td>
			<td>https://www.researchgate.net/publication/273383414_54sample_datamatrix_geneIDs_Fasoli2012</td>
		</tr>
		<tr>
			<td>Multi Experiment Viewer (MeV)</td>
			<td></td>
			<td></td>
			<td>http://mev.tm4.org/#/welcome</td>
		</tr>
		<tr>
			<td>Sequence Read Archive (SRA)</td>
			<td></td>
			<td></td>
			<td>https://www.ncbi.nlm.nih.gov/sra</td>
		</tr>
		<tr>
			<td>R</td>
			<td></td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>EMBOSS Needle (EMBL-EBI)</td>
			<td></td>
			<td></td>
			<td>http://www.ebi.ac.uk/Tools/psa/emboss_needle/</td>
		</tr>
	</tbody>
</table>

comprehensive workflow for the genome-wide identification and expression meta-analysis of the atl e3 ubiquitin ligase gene family in grapevine

Klassifizierung und Nomenklatur der Gene in einer Familie können erheblich zur Beschreibung von der Vielfalt der kodierten Proteine und die Vorhersage von Familienfeiern basierend auf einige Features, wie beispielsweise das Vorhandensein von Sequenzmotive oder bestimmten beitragen Standorte für Post-translationale Modifikation und das Expressionsprofil Familienmitglieder unter verschiedenen Bedingungen. Diese Arbeit beschreibt ein detailliertes Protokoll für gen-Familie-Charakterisierung. Hier wird das Verfahren bis hin zur Charakterisierung der Arabidopsis Tóxicos in Levadura (ATL) E3 Ubiquitin Ligase Familie in Grapevine angewendet. Die Methoden umfassen die genomweite Identifizierung von Familienangehörigen, die Charakterisierung des Gens Lokalisation, Struktur und Vervielfältigung, die Analyse der erhaltenen Protein Motive, die Vorhersage von Protein-Lokalisierung und Phosphorylierung-Sites sowie gen Expression profiling innerhalb der Familie in verschiedenen Datensätzen. Solches Verfahren, die zu weiteren Analysen je nach experimentellen Zwecken verlängert werden konnten, angewandt werden, für jede Genfamilie in alle Pflanzenarten für die genomische Daten verfügbar sind, und es liefert wertvolle Informationen um interessante Kandidaten zu identifizieren für funktionelle Studien geben Einblicke in die molekularen Mechanismen der Pflanze Anpassung an ihre Umwelt.

Das VIT_05s0077g01970-gen, als am ähnlichsten A. Thaliana ATL2 (At3g16720) durch eine BLASTp Suche, diente als Sonde um die ATL Familienmitglieder in das Genom der Weinrebe Umfrage identifiziert (V. Vinifera cv Pinot Noir PN40024). Die PSI-BLAST-Analyse kamen nach ein paar Zyklen zeigt eine Liste der vermeintlichen Gene aus der Weinrebe ATL Genfamilie (Abbildung 1A). Das Vorhandensein der kanonischen RING-H2-Domäne für jeden Kandidaten wu...

Watch this Scientific Journal Video about Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine at JoVE.com

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Dieser Artikel beschreibt das Verfahren für die Identifizierung und Charakterisierung einer Genfamilie in Grapevine, die zur Familie der Arabidopsis Tóxicos in Levadura (ATL) E3 Ubiquitin Ligases angewendet.

Umfassenden Workflow zur genomweiten Identifizierung und Ausdruck Meta-Analyse von der ATL E3 Ubiquitin Ligase Genfamilie in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

comprehensive-workflow-for-genome-wide-identification-expression-meta

Research

JoVE Journal

Biology

10.4K Aufrufe.  Università degli Studi di Verona. Dieser Artikel beschreibt das Verfahren für die Identifizierung und Charakterisierung einer Genfamilie in Grapevine, die zur Familie der Arabidopsis Tóxicos in Levadura (ATL) E3 Ubiquitin Ligases angewendet.

Serie: Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

Die Herstellung von primären hämatopoetischen Zellkulturen aus Knochenmark der Maus für die Elektroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Forschung

Biologie

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

Spinal Cord Elektrophysiologie

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Mit dem Gene Pulser MXcell Elektroporation System zur Primary Cells mit hohem Wirkungsgrad zu transfizieren

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robotik und Dynamic Image Analysis für Studies of Gene Expression in pflanzlichen Geweben

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

Verwendung eines automatisierten Cell Counter zu Genexpressionsstudien Simplify: siRNA Knockdown von IL-4 abhängige Genexpression in Namalwalzellen

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Genomweite Analyse mittels ChIP zu Isoform-spezifische Gene-Targets zu identifizieren

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Rekonstitution der Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

Transposon mutagenesis and single-gene deletion are two methods applied in genome-wide gene knockout in bacteria 1,2. Although transposon mutagenesis is less time consuming, less costly, and does not require completed genome information, there are two weaknesses in this method: (1) the possibility of a disparate mutants in the mixed mutant library that counter-selects mutants with decreased competition; and (2) the possibility of partial gene inactivation whereby genes do not entirely lose their function following the insertion of a transposon. Single-gene deletion analysis may compensate for the drawbacks associated with transposon mutagenesis. To improve the efficiency of genome-wide single gene deletion, we attempt to establish a high-throughput technique for genome-wide single gene deletion using Streptococcus sanguinis as a model organism. Each gene deletion construct in S. sanguinis genome is designed to comprise 1-kb upstream of the targeted gene, the aphA-3 gene, encoding kanamycin resistance protein, and 1-kb downstream of the targeted gene. Three sets of primers F1/R1, F2/R2, and F3/R3, respectively, are designed and synthesized in a 96-well plate format for PCR-amplifications of those three components of each deletion construct. Primers R1 and F3 contain 25-bp sequences that are complementary to regions of the aphA-3 gene at their 5' end. A large scale PCR amplification of the aphA-3 gene is performed once for creating all single-gene deletion constructs. The promoter of aphA-3 gene is initially excluded to minimize the potential polar effect of kanamycin cassette. To create the gene deletion constructs, high-throughput PCR amplification and purification are performed in a 96-well plate format. A linear recombinant PCR amplicon for each gene deletion will be made up through four PCR reactions using high-fidelity DNA polymerase. The initial exponential growth phase of S. sanguinis cultured in Todd Hewitt broth supplemented with 2.5% inactivated horse serum is used to increase competence for the transformation of PCR-recombinant constructs. Under this condition, up to 20% of S. sanguinis cells can be transformed using ~50 ng of DNA. Based on this approach, 2,048 mutants with single-gene deletion were ultimately obtained from the 2,270 genes in S. sanguinis excluding four gene ORFs contained entirely within other ORFs in S. sanguinis SK36 and 218 potential essential genes. The technique on creating gene deletion constructs is high throughput and could be easy to use in genome-wide single gene deletions for any transformable bacteria.

Genome-wide Gene Deletions in Streptococcus sanguinis by High Throughput PCR

An efficient genome-wide single gene mutation method has been established using Streptococcus sanguinis as a model organism. This method has achieved via high throughput recombinant PCRs and transformations.

Genomweiten Gen-Deletionen in<em&gt; Streptococcus sanguinis</em&gt; Von High Throughput PCR

Transposon  mutagenesis and single-gene deletion are two methods applied in genome-wide  gene knockout in bacteria 1,2. Although transposon mutagenesis is ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA Screening zu Ubiquitin und Ubiquitin-ähnlichen System Regler der biologischen Stoffwechselwege in kultivierten Säugerzellen identifizieren

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...