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. 1977 Aug;74(8):3297–3301. doi: 10.1073/pnas.74.8.3297

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: Nucleosomal proteins associated with a highly repetitive mammalian DNA

Phillip R Musich 1, Fred L Brown 1, Joseph J Maio 1
PMCID: PMC431537  PMID: 269392

Abstract

Component α DNA is a homogeneous, highly repetitive fraction that comprises nearly a quarter of the African green monkey (Cercopithecus aethiops) genome. By restriction enzyme analysis, it has a repeat periodicity of 176 ± 4 nucleotide base pairs, corresponding closely with the length of DNA contained within a nucleosome. The sequence is organized into large blocks of constitutive heterochromatin. A method is described here for the isolation of intact polynucleosomal arrays containing only component α sequences. Isolated monkey nuclei are treated with EcoRI, which releases only component α nucleosomal arrays; the arrays are then fractionated and purified by sedimentation in sucrose gradients. The method permits a compositional analysis of the proteins associated with a constitutively repressed, heterochromatic sequence.

The major differences in the proteins associated with component α nucleosomes that distinguish them from the bulk DNA nucleosomes are a decrease in the content of the H1 histones in the component α nucleosomes and a concomitant increase in the amount of certain nonhistone proteins. The specific observations are: (i) In the component α nucleosomes, 65-70% of the proteins were nonhistone proteins; this contrasts with the value, 40%, for nonhistone proteins associated with nucleosomes containing bulk DNA. (ii) The amount of H1 histone in chromatin containing predominantly bulk DNA was about 13.7%. However, the H1 histone was depleted and possibly absent in component α oligonucleosomes. (iii) Coincident with the decrease in the H1 histones and in the same molecular weight range (24,000-43,000), there appeared five minor nonhistone proteins. The minor, low-molecular-weight, nonhistone proteins were not detected in chromatin containing bulk DNA but they represented nearly 12% of the protein in component α nucleosomes. The resistance to salt extraction (0.6-2.0 M NaCl) indicates that the low-molecular-weight nonhistone proteins are tenaciously bound to the component α nucleosomes. In addition, a class of high-molecular-weight (>100,000) nonhistone proteins was enriched 5- or 6-fold in component α oligonucleosomes. The relative amounts of the nucleosome core histones were not changed.

Keywords: EcoRI restriction endonuclease, nucleosome fractionation, mammalian repetitive DNA sequences, histone and nonhistone proteins

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Selected References

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