Ringraziamo il Dott. Patricia Manosalva per le sue preziose informazioni sul manoscritto. Il finanziamento è stato fornito dal Programma Nazionale Genoma Plant Science Foundation, il numero di riconoscimento DBI-0605059.

Parte 1: Materiale vegetale N. benthamiana piante utilizzate per tacere dovrebbe essere intorno al 2 ½ settimane di vita che è il momento in cui i cotiledoni e le prime 2 - 4 foglie vere sono emerse. Pomodoro (Solanum lycopersicum) piante vengono utilizzate 7-8 giorni dopo l&#39;emergenza, quando le foglie vere non sono ancora apparsi. Parte 2: VIGS GIORNO 1 <ol><li> Per ogni esperimento, Agrobacterium tumefaciens ospitare pTRV1, pTRV2, pTRV2-Pds e pTRV2-host gene bersaglio vengono coltivati ​​su piastre di agar LB integrata con 50 mg / ml di kanamicina e 100 mg / ml di rifampicina. La kanamicina seleziona per la pTRV plasmide mentre la rifampicina lo fa per l&#39;Agrobacterium. Incubare la piastra a 30 ° C per 2 giorni. Silenziamento del PDS causerà le piante a photobleach ed è utilizzato come controllo di efficienza a tacere. Inoltre, i geni per essere inibiti deve essere clonato in un vettore pTRV2. C&#39;è un gateway compatibile pTRV2 vettore che possono facilitare la clonazione ed è descritto da Liu et al. (2002). </li></ol> 3 ° GIORNO <ol><li> Inoculare 2-3 ml di coltura liquida LB con gli antibiotici di cui sopra per ciascuno dei ceppi. Incubare agitando a 30 ° C per 16 - 18 ore e 200 giri al minuto </li></ol> GIORNO 4 <ol><li> Inoculare 1: 25 diluizione della coltura principale in una media secondaria di induzione liquido (IM) cultura IM con kanamicina, rifampicina e 200 acetosyringone mM (Tabelle 1, 2 e 3). Il acetosyringone viene utilizzato come un induttore dei geni vir di Agrobacterium che sono necessarie per T-DNA trasferimento nella pianta 9, mentre l&#39;IM imita l&#39;ambiente questo patogeno incontra nel apoplast ospitante. Incubare agitando a 30 ° C per 20 - 24 ore e 200 giri al minuto </li></ol> GIORNO 5 <ol><li> Raccogliere le cellule per centrifugazione per 10 minuti a 3000 x g. Risospendere nello stesso volume che la cultura originale era con 10 mM MgCl 2, 10 mM MES pH 5,5. Cellule possono essere delicatamente in agitazione per sospendere di nuovo loro. </li><li> Centrifugue le cellule di nuovo per 10 minuti a 3000 x g. Risospendere in metà del volume della cultura di origine con 10 mM MgCl2, 10 mM MES pH 5,5. </li><li> Preparare una sospensione batterica con un diametro esterno 600 di 0,3 per ogni coltura batterica. Aggiungi acetosyringone ad una concentrazione finale di 400 mM alla cultura pTRV1. </li><li> Mescolare le culture che contiene il pTRV1 e pTRV2 (o il pTRV2 contenente il gene di interesse) in un rapporto 1 a 1. Includono anche una pTRV2-PDS controllo. Si prega di notare che la concentrazione acetosyringone finale è ora 200 micron e che ogni cultura è a 600 OD di 0,15. </li><li> Etichetta le piantine di essere infiltrati con il gene per essere messa a tacere e la data dell&#39;esperimento. </li><li> Fare un buco in ogni foglia di essere infiltrati con un ago. Utilizzare una siringa da 1 ml manco a infiltrarsi nella sospensione batterica nelle piantine. Per il pomodoro, si infiltrano entrambi i cotiledoni mentre per N. benthamiana, infiltrare il più grande due foglie vere. Cinque mL di ciascuna miscela batterica dovrebbe essere sufficiente per infiltrarsi 15 N. benthamiana e 25 piantine di pomodoro. Evitare la contaminazione incrociata, cambiando i guanti tra infiltrazioni e non innaffiare le piante fino al giorno successivo, dopo l&#39;inoculazione. </li><li> Gli impianti sono tenuti a 20-22 ° C in una camera di crescita con una lunghezza del giorno 16 ore e 50% UR per almeno 3 settimane e mezzo prima di poter essere utilizzato per le analisi. </li></ol> Parte 3: I risultati rappresentativi La Figura 1 mostra un esperimento rappresentativo con N. benthamiana e piante di pomodoro a tacere per PDS. Le piante mostrano il fenotipo caratteristico photobleaching osservato in impianti con diminuita quantità di carotenoidi. Per il PDS-tacere le piante di controllo, inizia photobleaching di essere visto non appena 1 ½ settimane dopo l&#39;infiltrazione. <img src="/files/ftp_upload/1292/Figure 1A 1292.jpg" alt="figura 1a" /><img src="/files/ftp_upload/1292/Figure 1B 1292.jpg" alt="figura 1b" /> Figura 1. Silenziamento del gene di controllo PDS cause photobleaching in N. benthamiana (A) e pomodoro (B) le piante. Fotografie sono state scattate 3 settimane e mezzo dopo tacere. Tabella 1. Preparazione del mezzo di induzione (IM). <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 400 mL </td><td> H 2 O distillata </td></tr><tr><td> 4,88 g. </td><td> MES (2 - (4 morfolino)-etano solfonico) </td></tr><tr><td> 2,5 g </td><td> Glucosio </td></tr><tr><td> 0,12 g </td><td> NaH 2 PO 4 </td></tr></tbody></table> Portare ad un volume finale di 475 ml con dH 2 O e portare il pH a 5,6. Autoclave. Dopo che il medium è raffreddato, aggiungere 25 ml di 20X sali AB. Tabella 2. Preparazione di sali AB. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 20 g </td><td> NH 4 Cl </td></tr><tr><td> 6 g </td><td> MgSO 4 · 7H 2 O </td></tr><tr><td> 3 g </td><td> KCl </td></tr><tr><td> 0,2 g </td><td> CaCl 2 </td></tr><tr><td> 0,05 g </td><td> FeSO 4 · 7H 2 O </td></tr></tbody></table> Portare ad un volume finale di 1 litro con acqua distillata. Autoclave. Essere consapevoli del fatto che i sali di AB precipitare come una polvere arancione. Basta mescolare bene agitando prima del loro utilizzo. Tabella 3. Preparazione di 200 Acetosyringone mM. Si prega di notare che acetosyringone deve essere preparato il giorno in cui verrà utilizzato. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> 19,6 mg </td><td> Acetosyringone (3 &#39;, 5&#39;-Dimethoxy-4&#39;-hydroxyacetophenone) </td></tr><tr><td> 500 microlitri </td><td> DMSO (dimetil solfossido) </td></tr></tbody></table>

<ol>
	<li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+as+a+plant+immune+system+against+viruses.">RNA silencing as a plant immune system against viruses.</a> Trends Genet. 17, 449-459 (2001).</li><li>Carrington, J. C., Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+microRNAs+in+plant+and+animal+development.">Role of microRNAs in plant and animal development.</a> Science. 301, 336-338 (2003).</li><li>Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microRNA+as+a+translational+repressor+of+APETALA2+in+Arabidopsis+flower+development.">A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development.</a> Science. 303, 2022-2025 (2004).</li><li>Palatnik, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+leaf+morphogenesis+by+microRNAs.">Control of leaf morphogenesis by microRNAs.</a> Nature. 425, 257-263 (2003).</li><li>Brigneti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+pathogenicity+determinants+are+suppressors+of+transgene+silencing+in+Nicotiana+benthamiana.">Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana.</a> EMBO J. 17, 6739-6746 (1998).</li><li>Liu, Y., Schiff, M., Dinesh-Kumar, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-induced+gene+silencing+in+tomato.">Virus-induced gene silencing in tomato.</a> Plant Journal. 31, 777-786 (2002).</li><li>MacFarlane, 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=Molecular+biology+of+the+tobraviruses.">Molecular biology of the tobraviruses.</a> J. Gen. Virol. 80, 2799-2807 (1999).</li><li>Senthil-Kumar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+study+to+determine+the+extent+of+gene+silencing+in+Nicotiana+benthamiana+and+other+Solanaceae+species+when+heterologous+gene+sequences+are+used+for+virus-induced+gene+silencing.">A systematic study to determine the extent of gene silencing in Nicotiana benthamiana and other Solanaceae species when heterologous gene sequences are used for virus-induced gene silencing.</a> New Phytol. 176, 782-791 (2007).</li><li>McCullen, C. A., Binns, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium+tumefaciens+and+plant+cell+interactions+and+activities+required+for+interkingdom+macromolecular+transfer.">Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer.</a> Annu. Rev. Cell Dev. Biol. 22, 101-127 (2006).</li><li>Rosebrock, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+E3+ubiquitin+ligase+targets+a+host+protein+kinase+to+disrupt+plant+immunity.">A bacterial E3 ubiquitin ligase targets a host protein kinase to disrupt plant immunity.</a> Nature. 448, 370-374 (2007).</li><li>Miki, D., Itoh, R., Shimamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+of+single+and+multiple+members+in+a+gene+family+of+rice.">RNA silencing of single and multiple members in a gene family of rice.</a> Plant Physiol. 138, 1903-1913 (2005).</li><li>Voinnet, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use,+tolerance+and+avoidance+of+amplified+RNA+silencing+by+plants.">Use, tolerance and avoidance of amplified RNA silencing by plants.</a> Trends Plant Sci. 13, 317-328 (2008).</li><li>Ekengren, S. K., Liu, Y., Schiff, M., Kumar-Dinesh, S. P., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+MAPK+cascades,+NPR1,+and+TGA+transcription+factors+play+a+role+in+Pto-mediated+disease+resistance+in+tomato.">Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato.</a> Plant J. 36, 905-917 (2003).</li><li>Liu, E., Page, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+cDNA+libraries+for+virus-induced+gene+silencing+(VIGS)+using+tobacco+rattle+virus.">Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus.</a> Plant. Methods. 4, 5-5 (2008).</li></ol>

Virale silenziamento genico è un metodo che permette un rapido schermi della genetica inversa. Evita la generazione di T-DNA o geni mediata trasposone knock-out, che sono disponibili solo in alcune piante come Arabidopsis e mais. Si aggira anche il processo che richiede tempo di trasformazione di piante e permette il targeting dei geni multipli allo stesso tempo, a condizione che abbiano homology sufficienti 10 o che diverse sequenze di host di destinazione sono disposti in tandem nel silenzio Vetto...

virus-induced gene silencing (vigs) in nicotiana benthamiana and tomato

Interferenza dell&#39;RNA (RNAi) è altamente specifico silenziamento genico fenomeno innescato da dsRNA 1. Questo meccanismo di silenziamento utilizza due grandi classi di RNA regolatori: microRNA, che sono prodotte da geni che codificano proteine ​​e non breve RNA interferenti (siRNA). Le piante usano RNAi per controllare trasposoni e di esercitare uno stretto controllo sui processi di sviluppo come la formazione di organi di fiori e foglie sviluppo 2,3,4. Piante anche usare RNAi per difendersi contro le infezioni da virus. Di conseguenza, molti virus si sono evoluti soppressori di silenziamento genico per consentire loro colonizzazione successo del loro ospite 5. Virale silenziamento genico (VIGS) è un metodo che sfrutta la RNAi mediato da meccanismi di difesa piante antivirali. In piante infette da virus modificato il meccanismo è specificamente mirate contro il genoma virale. Tuttavia, con vettori di virus che trasportano sequenze derivate da geni dell&#39;organismo ospite, il processo può essere ulteriormente mirate contro il mRNA host corrispondente. VIGS è stato adattato per la genomica funzionale high-throughput nelle piante utilizzando il patogeno per le piante Agrobacterium tumefaciens per fornire, tramite il suo Ti plasmide, un virus ricombinante portando l&#39;intero o parte della sequenza genica mirata per tacere. Diffusione del virus sistemico e il endogeno impianti RNAi macchinari prendersi cura del resto. dsRNAs corrispondente al gene bersaglio vengono prodotti e poi scisso dal Dicer ribonucleasi in siRNA del 21-24 nucleotidi di lunghezza. Queste siRNA in ultima analisi, guida l&#39;RNA di silenziamento indotto complesso (RISC) a degradare la trascrizione obiettivo 2. Diversi vettori sono stati impiegati in VIGS e uno dei più frequentemente utilizzato è basato su tabacco sonaglio virus (TRV). TRV è un virus bipartito e, come tale, A. due diversi ceppi di Agrobacterium sono usati per VIGS. Si porta pTRV1, che codifica le funzioni di replicazione virale e il movimento, mentre l&#39;altro, pTRV2, porti la proteina di rivestimento e la sequenza usata per VIGS 6,7. Inoculazione di Nicotiana benthamiana e di piantine di pomodoro con un misto di entrambi i risultati ceppi di silenziamento genico. Silenziamento del endogeno phytoene desaturasi (PDS) del gene, che provoca photobleaching, viene utilizzato come controllo per l&#39;efficienza VIGS. Va notato, tuttavia, che il silenziamento di pomodoro è generalmente meno efficiente rispetto a N. benthamiana. Abbondanza trascrizione dell&#39;RNA del gene di interesse deve essere sempre misurata per garantire che il gene bersaglio è stato efficacemente down-regolato. Tuttavia, sequenze di geni eterologhi da N. benthamiana può essere utilizzato per mettere a tacere i rispettivi ortologhi di pomodoro e viceversa 8.

Watch this Scientific Journal Video about Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato at JoVE.com

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Descrizione di un virus-indotto silenziamento genico (VIGS) metodo per knock-down di espressione genica in<em&gt; Nicotiana benthamiana</em&gt; E pomodoro.

Virale silenziamento genico (VIGS) in Nicotiana benthamiana E Pomodoro

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

virus-induced-gene-silencing-vigs-in-nicotiana-benthamiana-and-tomato

Research

JoVE Journal

Biology

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

52.2K Views.  Cornell University. Descrizione di un virus-indotto silenziamento genico (VIGS) metodo per knock-down di espressione genica in Nicotiana benthamiana E pomodoro.

Video: Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Vivere Imaging calcio cella Combinato con il silenziamento del gene mediata siRNA Identifica Ca 2 + Canali di perdite nella membrana ER e nei relativi meccanismi regolatori

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Ricerca

Biologia

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Induzione e test di ipossia in coltura cellulare

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

Mediata da Agrobacterium Virus-Induced tacere Assay Gene In Cotone

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

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.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo mRNA

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

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

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

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

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

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

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltrazione e Agroinfezione PVX in Benthamiana di Patate e Nicotiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

Ottimizzazione e utilizzo di<em&gt; Agrobacterium</em&gt;-Mediata transitoria produzione di proteine ​​in<em&gt; Nicotiana</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

Chitosan/Interfering RNA Nanoparticle Mediated Gene Silencing in Disease Vector Mosquito Larvae

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field.

Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae.

Il chitosano / interferenti RNA nanoparticelle mediata silenziamento genico in Disease Vector Mosquito Larve

Vector mosquitoes inflict more human suffering than any other organism&#8212;and kill more than one million people each year. The mosquito genome projects ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Meiotic mandrino valutazione in mouse Ovociti mettendo a tacere siRNA-mediata

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Analisi di Imaging di complementazione di luciferase in foglie di Nicotiana benthamiana per transitoriamente determinare dinamiche di interazione proteina-proteina

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...