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High-resolution fractionation of nucleosomes: minor particles, "whiskers," and separation of mononucleosomes containing and lacking A24 semihistone

Levinger, Louis and Varshavsky, Alexander (1980) High-resolution fractionation of nucleosomes: minor particles, "whiskers," and separation of mononucleosomes containing and lacking A24 semihistone. Proceedings of the National Academy of Sciences of the United States of America, 77 (6). pp. 3244-3248. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:LEVpnas80

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Abstract

Staphylococcal nuclease digests of HeLa chromatin fractionated on low ionic strength nucleoprotein gels have been further analyzed by second-dimension DNA and protein gel electrophoresis. In vivo radioactive labeling of chromatin components and use of longer gels allowed a higher sensitivity and resolution than has been previously reported for this approach. A number of nonhistone protein spots and about 20 DNA spots can be detected in the mononucleosomal region of the second-dimension gel. In particular, there are three DNA spots identical in DNA size that correspond to three discrete kinds of core mononucleosomes resolved on the first-dimension nucleoprotein gel. Analysis of protein composition shows that the most rapidly migrating particle contains all four core histones but no A24 semihistone (A24 is a covalent conjugate of histone H2A and a specific nonhistone protein, ubiquitin), whereas the other two core mononucleosomes contain A24 semihistone. Thus, one can now quantitatively separate the A24-lacking core mononucleosomes from those containing A24, making it possible to directly address the question of whether A24 is associated with nucleosomes containing a specific subset of DNA sequences. Additional features of two-dimensional nucleoprotein-DNA patterns are "whiskers," which run slower than core mononucleosomes in the nucleoprotein dimension and both faster and slower than core-length DNA in the DNA dimension. In more extensive digests, "secondary whiskers" are observed, which run faster than core mononucleosomes in both dimensions and appear to coincide with previously described subnucleosomal particles SN7 and SN8 [Bakayev, V., Bakayeva, T. & Varshavsky, A. (1977) Cell 11, 619-629]. Possible mechanisms of whisker formation are discussed.


Item Type:Article
Additional Information:Copyright © 1980 by the National Academy of Sciences. Communicated by Gary Felsenfeld, March 13, 1980. We thank Frederick Boyce and Bruce Yazejian for their collaboration at early stages of this work and Jim Barsoum for helpful discussions. These studies were supported by grants to A.V. from the National Institutes of Health (GM26086) and the Whittaker Health Sciences Fund. L.L. is supported by a National Institutes of Health postdoctoral fellowship (AI 5937). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Subject Keywords:nuclease digestion, nucleoprotein gel electrophoresis, two-dimensional fractionation
Record Number:CaltechAUTHORS:LEVpnas80
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:LEVpnas80
Alternative URL:http://www.pnas.org/cgi/content/abstract/77/6/3244
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1366
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Jan 2006
Last Modified:26 Dec 2012 08:44

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