Gli autori desiderano ringraziare Drs. Philina S. Lee e Alan D. Grossman per la formazione inizialmente LAS in microscopia a fluorescenza. Gli autori hanno anche ringraziare Drs. Melanie Berkmen e Hajime Kobayashi aiuto e suggerimenti per l&#39;imaging. Questo lavoro è stato supportato da start-up fondi del Collegio di Letteratura, Scienze e Arti e dal Dipartimento di Biologia Molecolare, Cellulare e dello Sviluppo presso l&#39;Università del Michigan.

Colture di cellule in crescita per microscopia 1. Uno o due giorni prima di imaging, preparare la B. subtilis ceppo contenente la proteina di fusione traslazionale che si desidera visualizzare. Turni di prova di 1A e 1B passi saranno necessari per determinare quale condizione di crescita fornisce le migliori immagini del vostro sforzo. Nella maggior parte dei casi, le cellule dovrebbero essere esposte durante la fase di crescita esponenziale. <ol class="ualpha"><li> Per alcune B. ceppi subtilis (in particolare, i ceppi che crescono poco, grazie all&#39;integrazione della fusione traslazionale tra il gene di interesse e GFP al suo locus endogena), la crescita iniziale per due giorni precedenti l&#39;imaging è necessario per immagini di alta qualità. Il primo giorno, striscia la B. subtilis ceppo di essere ripreso su agar LB con antibiotico selettivo (s) e incubare una notte a 30 ° C. Il secondo giorno, inoculare 100 l di soluzione salina allo 0,85% con una singola colonia ed eseguire tre diluizioni di 10 volte seriale su piastre di agar LB (con antibiotici selettivi), placcatura 100 l di ogni diluizione, seguita dalla notte di incubazione a 30 ° C. Il terzo giorno, selezionare la piastra di diluizione con luce crescita confluente di cellule. Luce crescita confluente su un piatto fornirà il terreno di coltura con una sana e in crescita esponenziale inoculo starter per produrre potenzialmente ottimi risultati di imaging (vedi punto 4). Questa fase è particolarmente utile per l&#39;imaging di altissima qualità a membrana con la macchia FM4-64. </li><li> Altri B. ceppi subtilis possono semplicemente essere applicate a terreno selettivo agarizzato LB per singole colonie con un bastone sterile il giorno prima di imaging e incubare una notte a 30 ° C. Il giorno seguente, le celle su questa piastra sarà sufficiente utilizzare come inoculo per l&#39;imaging (vedi punto 4). NOTA: per B. subtilis, culture durante la notte in mezzo liquido non sono adatti a causa di una combinazione di formazione di spore e le risposte del quorum, che farà un lungo periodo di crescita ritardo di fase 1. </li></ol> 2. La mattina di imaging versare 10 ml di S7 50 media 1-3 in un pallone da 125 ml Erlenmeyer sterile. 3. Risospendere le cellule in un massimo di 2 ml di S7 50 di media dalla piastra LB agar con singole colonie o le cellule confluenti luce per ottenere un inoculo. 4. Aggiungi sufficiente S7 50 medio-cell inoculo per la beuta contenente 10 ml S7 50 di media per una cultura con una densità iniziale ottica misurata a 600 nm (OD 600) di ~ 0,08-0,1. A questo punto è importante avere almeno 10 volte l&#39;aria al rapporto medio. Quindi, utilizziamo 10-12,5 mL di terreno inoculato in un pallone di 125 ml Erlenmeyer. 5. Crescono ogni cultura a 30 ° C fino a quando la cultura raggiunge un diametro esterno 600 ~ 0,5-0,8. Bagni di acqua agitazione sono da preferire con giri al minuto da 150-200. Culture sfidato con gli agenti che danneggiano il DNA inducendo o mancata corrispondenza dovrebbe essere in una fase iniziale di crescita esponenziale OD 600 in modo tale che le cellule raggiungerà l&#39;obiettivo OD 600 con l&#39;ulteriore periodo di crescita che è necessario per il trattamento (vedi tabella 1) Per esempio, 2-aminopurine richiede un&#39;ora di trattamento, e quindi una cultura trattati con 2-aminopurine dovrebbe essere di 0,3-0,4 OD 600 ~, tale che l&#39;un&#39;ora di trattamento / risultati di crescita in un OD 600 ~ 0,5-0,8, per cui le cellule sono pronte per l&#39;imaging. Preparazione del campione 1. Pipettare 300 ul di cellule in coltura in una provetta per microcentrifuga da 1,5 ml. 2. Se l&#39;imaging delle membrane cellulari si desidera, aggiungere 1:1000 diluizione di FM4-64 membrana macchia (Invitrogen) per ogni campione da un mg / 1 ml soluzione madre. Una titolazione di FM4-64 può essere richiesto per ottenere il segnale di fluorescenza desiderato. Una gamma titolazione tipica è 1:100, 1:1000 e 1:10.000. 3. Lasciare i campioni a temperatura ambiente fino a una decina di minuti, mentre la diapositiva si sta preparando con 1% di agarosio in 1X medio Spizizens come descritto di seguito (vedi punto 1 di &quot;preparazione del vetrino&quot;). 4. Se la concentrazione di cellule è richiesto, noi preferiamo celle filtranti con filtro da 0,2 micron con un apparato vuoto. Le celle possono poi essere lavati delicatamente dalla superficie del filtro con PBS, un altro tampone appropriato o medio prima di visualizzazione. Far scorrere la preparazione 1. Preparare l&#39;1% di agarosio con 1X Spizizens. Aggiungere 5 ml di 10X magazzino Spizizens a 45 ml ddH2O con 0,5 g di agarosio, e forno a microonde a sciogliersi. Agarosio fuso a caldo è difficile da pipetta, e solidificato agarosio non entrerà la punta della pipetta. L&#39;agarosio devono essere equilibrati in un bagno d&#39;acqua ad una temperatura di circa 55-65 ° C, per entrare la punta della pipetta senza solidificazione all&#39;interno del<html> punta. 2. Disegna 20 l di Spizizens con 1% di agarosio fuso nella punta della pipetta. Pipetta una goccia (circa 1-2 mL) della soluzione di agarosio in ogni pozzetto (usiamo pure 15-diapositive, vedi Tabella 2) per formare superficiale pastiglie agarosio. L&#39;agarosio si solidificano immediatamente. Le diapositive devono essere fatte immediatamente prima dell&#39;applicazione delle cellule. Se le pastiglie di agarosio vengono lasciati a temperatura ambiente da soli, possono disidratarsi in pochi minuti e diventano inutilizzabili. Pastiglie agarosio deve essere centrato e riempire ogni bene, ma hanno l&#39;altezza minima per evitare di interrompere il coprioggetto, se le gocce sono troppo elevati (bubble-simili) o se il campione si estende al di là del confine bene, è sufficiente passare il bene pulire con un Kimwipe e applicare un agarosio nuovo pad. 3. Applicare l&#39;intero campione di 300 microlitri (vedi punto 6) alla diapositiva, distribuiti in cima ad ogni pad agarosio. Questa quantità di cellule dovrebbe essere sufficiente a coprire ogni pozzetto nella diapositiva. 4. Lasciare il vetrino caricato con celle a temperatura ambiente (23 ° C) per circa dieci minuti. Questo passo consentirà alle cellule di stabilirsi sul pad agarosio diventando immobile e pronto per l&#39;imaging. Per esigenze specifiche questa temperatura può avere bisogno di essere cambiato, per esempio, durante l&#39;esecuzione di esperimenti che richiedono un cambiamento di temperatura 4. 5. Aspirare via del mezzo in eccesso da ogni pozzetto senza rimuovere le pastiglie stesse. 6. Applicare il coprioggetto alla diapositiva, premendo con decisione e in modo uniforme, ma delicatamente, tutta l&#39;area della diapositiva. Verificare che la polizza di copertura è contro la diapositiva e che la polizza di copertura non viene sollevata sopra il vetrino. Visualizzazione di cellule 1. Molti microscopi a fluorescenza diversi funzionerà bene. E &#39;fondamentale avere un 100X lente immersione in olio. Il laboratorio utilizza Simmons: <ul><li> Olympus BX61 microscopio dotato di 1,45 lente NA TIRFM olio obiettivo 100X ad immersione </li><li> Hamamatsu ORCAR 2 telecamera CCD raffreddata </li><li> Lumen 200 arco in metallo (Prima) fonte di luce. </li><li> Per il rilevamento di GFP (CIC), filtro eccitazione ed emissione 460-500 510-560 </li><li> Per il rilevamento di FM4-64 (TRITC), filtro eccitazione ed emissione 510-560 572-648. </li><li> Le immagini sono state catturate usando SlideBook 4,2 (Figura 1) </li></ul> 2. Portare le cellule a fuoco con luce bianca. 3. Catturare l&#39;immagine con filtri appropriati per ciascun fluoroforo. Rappresentante Risultati Immagini rappresentative sono (Figura 1). GFP focolai devono essere ben definiti, mentre FM4-64 colorazione deve essere brillante e chiaro 4-7. Immagini GFP o membrana possono essere pseudo-colorato con qualsiasi colore, per consentire la massima qualità dell&#39;immagine che sarà presentato senza perdere alcun dato. <img src="/files/ftp_upload/1736/1736fig1.jpg" alt="Figura 1" /> Figura 1: le immagini GFP Rappresentante di B. subtilis proteine ​​di fusione traslazionale. proteine ​​GFP sono mostrati in verde, mentre FM4-64 membrana colorazione è mostrato in rosso. La barra di scala bianca indica 3 micron (A) muts-GFP [rilevanti genotipo: muts - GFP (SPC), amyE:: Pspac mutL (gatto)]. In presenza di 600 mg / ml 2-aminopurine e 1 mM IPTG. FM4-64 segnale non viene presentato per mostrare più chiaramente le foci muts-GFP (B) MutL-GFP [rilevanti genotipo: mutL:: mutL-GFP (SPC)].. In presenza di 2-aminopurine (C) DnaX-GFP [rilevanti genotipo: dnaX:: dnaX-GFP (SPC)]. (D) Reca-GFP [rilevanti genotipo: recA:: recA-GFP (SPC)] in presenza di 20 ng / mL mitomicina C. (E) TagC-GFP [rilevanti genotipo: tagC:: tagC-GFP (SPC) ] in presenza di 1 mg / mL mitomicina C. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Trattamento / concentrazione o importo </td><td> Funzione </td><td> Tempo di incubazione </td></tr><tr><td> Mitomicina C (MMC) [20 e 150 ng / ml] </td><td> DNA agente alchilante </td><td> 1 ora </td></tr><tr><td> 2-aminopurine (2-AP) [600 mg / ml] </td><td> mancata corrispondenza inducendo </td><td> 1 ora </td></tr><tr><td> Idrossiurea (HU) </td><td> esaurisce dNTP </td><td> 3 ore </td></tr><tr><td> luce ultravioletta (UV) 20 J/m2 </td><td> Timina-timina dimeri e 6-4 fotoprodotti </td><td> varia a seconda della fonte, di solito 20 secondi </td></tr><tr><td> grigi (radiazioni ionizzanti) da 5 a 100 Gy </td><td> Rompe doppio filamento, le interruzioni di singolo filamento e siti danno base </td><td> varia a seconda della fonte </td></tr></tbody></table>&quot;&gt; Tabella 1. Sintesi dei trattamenti che danneggiano il DNA e indurre mancata corrispondenza <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Multitest scivolo, 15-oltre </td><td> MP Biomedicals </td><td> 6041505E </td><td></td></tr><tr><td> Microscopio copertura in vetro </td><td> Pescatore </td><td> 12-544-B </td><td></td></tr><tr><td> Agarosio </td><td> Pescatore </td><td> BP160-500 </td><td></td></tr><tr><td> Mitomicina C </td><td> Sigma </td><td> M0503-2MG </td><td> mutageno, indossare guanti </td></tr><tr><td> 2-Aminopurine </td><td> Sigma </td><td> A3509-250mg </td><td> guanti </td></tr><tr><td> Idrossiurea </td><td> Sigma </td><td> H8627-25G </td><td> guanti </td></tr><tr><td> FM4-64 </td><td> Invitrogen </td><td> T13320 </td><td> sensibile alla luce </td></tr></tbody></table> Tabella 2. Lista dei reagenti e attrezzature specifiche:

<ol>
	<li>Hardwood, C. R., Cutting, S. 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> Molecular Biological Methods for Bacillus. , (1990).</li><li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle.">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis.">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis.">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li><li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis.">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li><li>Simmons, L. A. <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+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li><li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mismatch+repair+in+bacterial+cells.">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li></ol>

Tentativi ed errori sono necessari per trovare condizioni di esposizione per immagini di altissima qualità per ogni ceppo, troviamo che 1 millisecondo è appropriato per le immagini a luce bianca, mentre l&#39;esposizione da 100 a 2000 ms sono appropriati per GFP (FITC) e FM4-64 (TRITC ) le immagini. Tempo di esposizione variano a seconda delle apparecchiature di imaging utilizzate. Si consiglia l&#39;uso di un ceppo per 15 pozzetti vetrino da microscopio per il più semplice di imaging, come la qualità pad e la diffu...

Ricette soluzione specifica 1: S7 10x 50 sali 0,5 M MOPS 100 mM di solfato di ammonio 50 mM di potassio fosfato monobasico Filtro sterilizzare, e avvolgere S7 50 supporti in fogli prima di stoccaggio Metalli 100x 0,2 M MgCl 2 70 mM CaCl 2 5 mM MnCl 2 0.1 mM ZnCl 2 100 mg / ml tiamina HCl 2 mM HCl 0.5 mM FeCl 3 * dH 2 O a volume finale * FeCl 3 deve essere aggiunto all&#39;ultimo, per evitare la precipitazione. Dopo la sterilizzazione del filtro, avvolgere 100x soluzione in un foglio di metallo. S7 50 mezzi 1x S7 50 sali 1x metallo 1% di glucosio 0,1% glutammato 40 mcg / mL triptofano 40 mcg / mL Fenilalanina H 2 O distillata a volume finale Spizizens 10x (grammi / L) 151,4 solfato di ammonio mM (20 g / L) 803,8 mM di potassio fosfato monobasico (140 g / L) 440,9 mM di potassio fosfato bibasico (60g / L) 34,0 mM citrato di sodio (10 g / L) 16,6 mM MgSO 4 (2g / L) dH 2 O a volume finale Filtro sterilizzare

imaging mismatch repair and cellular responses to dna damage in bacillus subtilis

Sia procarioti ed eucarioti rispondere a danni al DNA attraverso una complessa serie di cambiamenti fisiologici. Alterazioni nell&#39;espressione genica, la redistribuzione delle proteine ​​esistenti e l&#39;assemblaggio di complessi proteici nuovo può essere stimolata da una varietà di lesioni del DNA e non corrispondenti coppie di basi del DNA. Microscopia a fluorescenza è stato utilizzato come un potente strumento sperimentale per visualizzare e quantificare le risposte a queste ed altre lesioni del DNA e per monitorare lo stato replicazione del DNA all&#39;interno della complessa architettura subcellulare di una cellula vivente. Fusioni traduzionali tra proteine ​​fluorescenti giornalista e componenti della replicazione del DNA e riparazione macchinari sono stati utilizzati per determinare i segnali che l&#39;obiettivo del DNA proteine ​​di riparazione delle lesioni ai loro affini<em&gt; In vivo</em&gt; E per capire come queste proteine ​​sono organizzati all&#39;interno delle cellule batteriche. Inoltre, fusioni trascrizionale e traslazionale legata ai promotori inducibili danni al DNA hanno rivelato che le cellule all&#39;interno di una popolazione hanno attivato le risposte allo stress genotossico. In questa recensione, forniamo un protocollo dettagliato per l&#39;utilizzo di microscopia a fluorescenza per l&#39;immagine del montaggio di riparazione del DNA e complessi di replicazione del DNA in singole cellule batteriche. In particolare, questo lavoro si concentra sulle proteine ​​mancata corrispondenza riparazione di imaging, ricombinazione omologa, replicazione del DNA e un SOS-inducibile proteina<em&gt; Bacillus subtilis</em&gt;. Tutte le procedure descritte qui sono facilmente suscettibili di complessi proteici di imaging in una varietà di specie batteriche.

Watch this Scientific Journal Video about Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis at JoVE.com

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Un protocollo dettagliato è descritto per l&#39;imaging in tempo reale la formazione di complessi di riparazione del DNA in<em&gt; Bacillus subtilis</em&gt; Cellule.

Imaging mismatch repair e risposte cellulari al danno del DNA in Bacillus subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

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Research

JoVE Journal

Biology

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

10.9K Views.  University of Michigan-Ann Arbor. Un protocollo dettagliato è descritto per l'imaging in tempo reale la formazione di complessi di riparazione del DNA in Bacillus subtilis Cellule.

Video: Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Due-e tridimensionale Live Cell Imaging di proteine ​​danno al DNA di risposta

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

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Biologia

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA arricchimento da una tecnologia basata trasposasi-NGS per l&#39;analisi della sequenza intera<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, e 9 geni coinvolti nella riparazione del DNA Damage

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Visualizzazione di miniSOG Tagged proteine ​​di riparazione del DNA, in combinazione con Electron spettroscopica Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Misurazione danno al DNA e ripristina in mouse Splenociti Dopo cronica<em&gt; In Vivo</em&gt; L&#39;esposizione a dosi molto basse di beta e gamma Radiazioni

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

In Vivo alcalina Comet Assay e l&#39;enzima modificato alcalina Comet Assay per la misurazione rotture del DNA Strand e danno ossidativo del DNA in fegato di ratto

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Indagando i dannosi effetti di basso pressione sterilizzazione al Plasma sulla sopravvivenza di Bacillus subtilis spore utilizzando Live Cell microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Analisi Microfluidic unicellulare del Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Visualizzazione e quantificazione dei danni al DNA specifici del sito basati sull'endonucleasi

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analisi della funzione E3 Ubiquitina ligasi

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...