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

Abstract

Component .alpha. 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 .alpha. sequences. Isolated monkey nuclei are treated with EcoRI, which releases only component .alpha. 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 .alpha. nucleosomes that distinguish them from the bulk DNA nucleosomes are a decrease in the content of the H1 histones in the component .alpha. nucleosomes and a concomitant increase in the amount of certain nonhistone proteins. In the component .alpha. nucleosomes, 65-70% of the proteins were nonhistone proteins; this contrasts with the value, 40%, for nonhistone proteins associated with nucleosomes containing bulk DNA. The amount of H1 histone in chromatin containing predominantly bulk DNA was about 13.7%. The H1 histone was depleted and possibly absent in component .alpha. oligonucleosomes. Coincident with the decrease in the H1 histones and in the same MW range (24,000-43,000) there appeared 5 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 .alpha. 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 .alpha. nucleosomes. A class of high molecular weight (> 100,000) nonhistone proteins was enriched 5- or 6-fold in component .alpha. oligonucleosomes. The relative amounts of the nucleosome core histones were not changed.