Name: assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning dna
Uploaded: 2013-09-10T17:00:00
Description: Watch this Scientific Journal Video about Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA at JoVE.com

Dette arbejde blev støttet af NIH tilskud GM45916 og GM66834 til JCH og et stipendium fra den internationale Rett Syndrom Foundation til AK Dette arbejde blev også støttet af NIH tilskud GM088409 og Howard Hughes Medical Institute bidrag til KL

1.. Montering af rekombinante Core Histoner ind octamerer Grundlag: Det første skridt i nukleosomal vifte rekonstituering er at forberede native kerne histon octamerer fra frysetørrede rekombinante kerne histoner. Histonproteinerne kombineres i lige store molære mængder og samles i histon octamerer ved dialyse af prøverne af et denaturerende puffer i genfoldningspuffer. <ol><li> Rense og lyofilisere rekombinante kerne histoner (H2A, H2B, H3, H4) som beskrevet 13.. </li><li>...

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Model nukleosomal arrays er et meget nyttigt redskab for in vitro-undersøgelse af kromatin struktur og funktion. For eksempel er de blevet bredt anvendt til at undersøge mekanismen for kromatin fiber kondensation i opløsning fra 30 til 34, og gjorde det muligt at finde en x-ray struktur af en tetranucleosome 35. For nylig har de vist sig nyttige i at decifrere de strukturelle effekter af specifikke kerne histon varianter, mutanter og posttranslationelle modifikationer 14-16,36.</su...

Eukaryote genomer ikke eksisterer som nøgent DNA, men snarere er komprimeret og organiseret af bundne proteiner. Disse komplekser af DNA og protein, som kaldes kromatin. Den grundlæggende gentagne enhed i kromatin er nucleosome. En nukleosom består af histon octamer og 146 basepar af DNA viklet omkring histon octameren omkring 1,6 gange 1. Histon octamer er sammensat af to eksemplarer hver af de centrale histoner H2A, H2B, H3 og H4. Core histon octamerer, der gentagne mellemrum langs en DNA-molekyle kaldes nukleosomal arrays. Den udvidede struktur nukleosomal arrays er blevet omtalt som den 10 nm fiber eller &quot;perler på en snor&quot;-struktur og er til stede in vitro under lave saltbetingelser 2. Den 10 nm fiber er i stand til at kondensere i højere orden strukturer gennem intra-matrix komprimering og / eller inter-matrix oligomerisation 2. Disse strukturer af højere orden kan induceres i nærvær af salte,eller kan påvirkes gennem binding af kromatin arkitektoniske proteiner til nukleosomal matrix 3,4. Niveauer af kromatin komprimering omvendt korreleret med hastigheden af transskription in vivo 5,6. Nyere forskning understreger betydningen af den strukturelle organisering af genomer i processer såsom differentiering, udviklingen af kræft, og andre 7,8. Brugen af ​​nukleosomal arrays til at studere kromatin struktur og funktion er blevet udbredt. Her beskriver vi en fremgangsmåde til samling af nukleosomal arrays fra rekombinante kerne histoner og nukleosom positionering DNA. Brug rekombinant DNA med tandemgentagelser af nukleosom positionering sekvenser giver mulighed for genoprettelse af arrays, som indeholder regelmæssigt fordelte nukleosomer. To af de mere populære positionering sekvenser er 5S rRNA-gensekvens og &quot;601&quot; sekvens 9,10. Den 601-sekvensen blev afledt af SELEX eksperimenter og more positionerer kraftigt nucleosomes end 5S sekvens 11. Følgelig linker-DNA længde 601 arrays er mere homogen. Tandemgentaget nukleosom positionering DNA opnås ved gelfiltrering 4,12. Rekombinante histoner oprenses fra E. coli under denaturerende betingelser 13. Anvendelsen af ​​rekombinante histoner tillader en at omhyggeligt at kontrollere histon sammensætning nukleosomal arrays. For eksempel kerne histoner bærer specifikke mutationer 14 eller post-translationelle modifikationer 15,16 kan erstatte vildtype kerne histoner. Sedimentation velocity eksperimenter overvåger hastigheden af sedimentering af makromolekyler i opløsning under en anvendt centrifugalkraft 17. Dette giver information om størrelsen og formen af ​​makromolekyler i en prøve. Sedimentation velocity eksperimenter er derfor et egnet værktøj til at studere løsning tilstandsændringer i kromatin fibergrund chromatinkondensering 18 struktur. Vigtigere er det, er det nødvendigt først at bruge sedimentationshastighed eksperimenter som en kvalitetskontrol skridt i nukleosomal matrix rekonstituering. Hvis DNA og nukleosomer ikke kombineres på rette molforholdet, kan arrays være under-eller over-mættet med kerne histoner. Således er oplysninger fra de sedimentationshastighed eksperimenter anvendes til at sikre, at DNA&#39;et er korrekt mættet med nukleosomer. Det er vigtigt at anvende alternative metoder til at estimere mætning af DNA med nukleosomer, især hvis du arbejder med en tidligere uncharacterized DNA-skabelon. Derfor har vi også beskrive en metode til analyse af nukleosomal arrays bruger atomic force mikroskopi (AFM). AFM er en kraftfuld teknik, der tillader visualisering af virkningerne af en række parametre, såsom graden af mætning, virkning af tilstedeværelsen af histon varianter eller virkninger af MgCl2 19,20. AFM har også været ansøged at studere nukleosom dynamik ved hjælp af tid bortfalder billeddannelse 21. In vitro samles nukleosomal 12-mer arrays er særligt modtagelige for AFM studier, fordi de hører til i den rigtige størrelse interval for AFM billeddannelse 22. I nærværende undersøgelse har vi brugt AFM af nukleosomal arrays som en kvalitetskontrol samt som et middel til at bekræfte data fra AUC (&quot;se er at tro&quot;). Ud over simple visualisering, AFM tillader måling af højde profiler af prøverne som en yderligere parameter.

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 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(3-Aminopropyl)triethoxysilane</td>
 <td>Sigma-Aldrich </td>
 <td>A3648-100ML</td>
 <td></td>
 </tr>
 <tr>
 <td>6-8 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>21-152-5</td>
 <td></td>
 </tr>
 <tr>
 <td>HiLoad Superdex 200 16/60 Column</td>
 <td>GE</td>
 <td>17-1069-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Vivaspin 50 kDa MWCO Centrifugal Concentrator</td>
 <td>Sartorius</td>
 <td>VS2031</td>
 <td></td>
 </tr>
 <tr>
 <td>12-14 kDa MWCO Dialysis Tubing</td>
 <td>Fisher</td>
 <td>08-667A</td>
 <td></td>
 </tr>
 <tr>
 <td>Illustra Sephacryl S-1000 Superfine</td>
 <td>GE</td>
 <td>17-0476-01 </td>
 <td></td>
 </tr>
 <tr>
 <td>XL-A/I Analytical Ultracentrifuge</td>
 <td>Beckman-Coulter</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning dna

Core histon octamerer, der gentagne mellemrum langs en DNA-molekyle kaldes nukleosomal arrays. Nukleosomal arrays opnås på en af to måder: oprensning fra in vivo-kilder eller rekonstitution in vitro fra rekombinante kerne histoner og tandemgentaget DNA nukleosom positionering. Sidstnævnte metode har den fordel, at til samling af en mere sammensætningsmæssigt ensartet og præcist placeret nukleosomal array. Sedimentationshastighed eksperimenter i den analytiske ultracentrifuge udbytte information om størrelse og form af makromolekyler ved at analysere den hastighed, hvormed de migrerer gennem opløsning under centrifugalkraft. Denne teknik, sammen med atomic force mikroskopi, kan anvendes til kvalitetskontrol, der sikrer, at størstedelen af ​​DNA-skabeloner er mættet med nukleosomer efter rekonstitution. Her beskriver vi de protokoller, der er nødvendige for at rekonstruere milligram mængder af længde og deres sammensætning defined nukleosomal arrays egnet til biokemiske og biofysiske studier af kromatin struktur og funktion.

For at illustrere den protokol, vi rekonstituerede nukleosomal arrays fra rekombinante Xenopus kerne histoner og DNA, der består af 12 tandem 207 bp gentagelser af 601 positioneringsforløbet (601 207 x 12). Vi først samles native octamerer fra lyofiliserede kerne histoner og renses de octamerer ved FPLC ved anvendelse af en S200 søjle (figur 1A). Større komplekser elueres tidligere fra S200 kolonne. Histoner generelt elueres i denne rækkefølge: uspecifikke histon aggregater, h...

Watch this Scientific Journal Video about Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA at JoVE.com

Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA

En metode præsenteres for rekonstituering af model nukleosomal arrays fra rekombinante kerne histoner og tandemgentaget DNA nukleosom positionering. Vi beskriver også, hvordan sedimentationshastighed eksperimenter i den analytiske ultracentrifuge og atomic force mikroskopi (AFM) anvendes til at overvåge omfanget af nukleosomal matrix mætning efter rekonstitution.

Montering af nukleosomal Arrays fra rekombinant Core Histoner og nukleosom Positioning DNA

Core histone octamers that are repetitively spaced along a DNA molecule  are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two  ...

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Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Isolation of Genomic DNA from Mouse Tails

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Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

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Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

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Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

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5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Design and Use of Multiplexed Chemostat Arrays

Design og brug af multiplekset kemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Effektiv Produktion og oprensning af rekombinant murin Kindlin-3 fra Insect Celler til Biofysiske Studies

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

Real-time Imaging af Single Engineered RNA-transkripter i levende celler Brug Ratiometrisk bimolekylær 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 ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Ikke-kromatografisk oprensning af rekombinant elastin-lignende polypeptider og deres fusioner med peptider og proteiner fra<em&gt; Escherichia coli</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...