Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome 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 ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. Th...
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| creator | Rogge, Ryan A. Kalashnikova, Anna A. Muthurajan, Uma M. Porter-Goff, Mary E. Luger, Karolin Hansen, Jeffrey C. |
| description | Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function. |
| doi_str_mv | 10.3791/50354 |
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Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.</description><subject>Cellular Biology</subject><subject>Chromatin - chemistry</subject><subject>DNA - chemistry</subject><subject>Histones - chemistry</subject><subject>Microscopy, Atomic Force - methods</subject><subject>Nucleosomes - chemistry</subject><subject>Ultracentrifugation - methods</subject><issn>1940-087X</issn><issn>1940-087X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpVkVtLAzEQhYMoXvsHfJC8CL5Uk82lyYtQ6hVERRR8EEJ2d1ZTdhNNdgv99662Vn2agfnOmeEMQgNKjtlI0xNBmOBraJtqToZEjZ7X__RbaCelKSEyI0Jtoq2MEyEFl9voZZwSNHk9x6HCt11RQ0ihsTUex2jnCVcxNPgBitDkzlvf4kmIgK9caoOHhK0vf1WA70NyrQve-Vd8djveQxuVrRMMlnUXPV2cP06uhjd3l9eT8c2wEDprhznRIEqdS6oBQJeSS14SS0mWV8oWgshClcJyxaRmLNNypCrKS6hUDpIwzXbR6cL3vcsbKAvwbbS1eY-usXFugnXm_8S7N_MaZoYpyTM56g2OlgYxfHSQWtO4VEBdWw-hS4ZyJhTXmtAePVygRQwpRahWaygxX58w35_ouYO_N62on-h7YH8BTMMMzDR00fcZLdWfBweM-g</recordid><startdate>2013</startdate><enddate>2013</enddate><creator>Rogge, Ryan A.</creator><creator>Kalashnikova, Anna A.</creator><creator>Muthurajan, Uma M.</creator><creator>Porter-Goff, Mary E.</creator><creator>Luger, Karolin</creator><creator>Hansen, Jeffrey C.</creator><general>MyJove Corporation</general><scope>ALOKQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>2013</creationdate><title>Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA</title><author>Rogge, Ryan A. ; Kalashnikova, Anna A. ; Muthurajan, Uma M. ; Porter-Goff, Mary E. ; Luger, Karolin ; Hansen, Jeffrey C.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c592t-b09e5d9b619eee9d6464d0a102bf8ac506c8d5a483693329678f14def8be60393</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Cellular Biology</topic><topic>Chromatin - chemistry</topic><topic>DNA - chemistry</topic><topic>Histones - chemistry</topic><topic>Microscopy, Atomic Force - methods</topic><topic>Nucleosomes - chemistry</topic><topic>Ultracentrifugation - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Rogge, Ryan A.</creatorcontrib><creatorcontrib>Kalashnikova, Anna A.</creatorcontrib><creatorcontrib>Muthurajan, Uma M.</creatorcontrib><creatorcontrib>Porter-Goff, Mary E.</creatorcontrib><creatorcontrib>Luger, Karolin</creatorcontrib><creatorcontrib>Hansen, Jeffrey C.</creatorcontrib><collection>JoVE Journal: Biology</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Journal of Visualized Experiments</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Rogge, Ryan A.</au><au>Kalashnikova, Anna A.</au><au>Muthurajan, Uma M.</au><au>Porter-Goff, Mary E.</au><au>Luger, Karolin</au><au>Hansen, Jeffrey C.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA</atitle><jtitle>Journal of Visualized Experiments</jtitle><addtitle>J Vis Exp</addtitle><date>2013</date><risdate>2013</risdate><issue>79</issue><issn>1940-087X</issn><eissn>1940-087X</eissn><abstract>Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. 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| source | Journal of Visualized Experiments : JoVE |
| subjects | Cellular Biology Chromatin - chemistry DNA - chemistry Histones - chemistry Microscopy, Atomic Force - methods Nucleosomes - chemistry Ultracentrifugation - methods |
| title | Assembly of Nucleosomal Arrays from Recombinant Core Histones and Nucleosome Positioning DNA |
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