Work

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

Year: 2013

Type: article

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. 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.

Source: Journal of Visualized Experiments

DOI: 10.3791/50354

Authors: Ryan A. Rogge, Anna A. Kalashnikova, Uma M. Muthurajan, Mary E. Porter-Goff, Karolin Luger, Jeffrey C. Hansen

Institution: Colorado State University

Language: English

Topic: Genomics and Chromatin Dynamics

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: bronze

Funders: Howard Hughes Medical Institute, International Rett Syndrome Foundation, National Institutes of Health, National Institute of General Medical Sciences