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. 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.
Biochemistry and Molecular Biology, Colorado State University.
Ryan A
RA
Rogge
Anna A
AA
Kalashnikova
Uma M
UM
Muthurajan
Mary E
ME
Porter-Goff
Karolin
K
Luger
Jeffrey C
JC
Hansen
D002843:Chromatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D006657:Histones / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods; D009707:Nucleosomes / Q000737:chemistry*; D014461:Ultracentrifugation / Q000379:methods
D002843:Chromatin; D006657:Histones; D009707:Nucleosomes; D004247:DNA