Nucleosome arrangement in green monkey α-satellite chromatin: Superimposition of non-random and apparently random patterns

Abstract

We have studied the structure of tandemly repetitive alpha-satellite chromatin (alpha-chromatin) in African green monkey cells (CV-1 line), using restriction endonucleases and staphylococcal nuclease as probes. While more than 80% of the 172-base-pair (bp) alpha-DNA repeats have a HindIII site, less than 15% of the alpha-DNA repeats have an EcoRI site, and most of the latter alpha-repeats are highly clustered within the CV-1 genome. EcoRI and HindIII solubilize approximately 8% and 2% of the alpha-chromatin, respectively, under the conditions used. EcoRI is thus approximately 30 times more effective than HindIII in solubilizing alpha-chromatin, with relation to the respective cutting frequencies of HindIII and EcoRI on alpha-DNA. EcoRI and HindIII solubilize largely non-overlapping subsets of alpha-chromatin. The DNA size distributions of both EcoRI- and HindIII-solubilized alpha-chromatin particles peak at alpha-monomers. These DNA size distributions are established early in digestion and remain strikingly constant throughout the digestion with either EcoRI or HindIII. Approximately one in every four of both EcoRI- and HindIII-solubilized alpha-chromatin particles is an alpha-monomer. Two-dimensional (deoxyribonucleoprotein leads to DNA) electrophoretic analysis of the EcoRI-solubilized, sucrose gradient-fractionated alpha-oligonucleosomes shows that they do not contain "hidden" EcoRI cuts. Moreover, although the EcoRI-solubilized alpha-oligonucleosomes contain one EcoRI site in every 172-bp alpha-DNA repeat, they are completely resistant to redigestion with EcoRI. This striking difference between the EcoRI-accessible EcoRI sites flanking an EcoRI-solubilized alpha-oligonucleosome and completely EcoRI-resistant internal EcoRI sites in the same alpha-oligonucleosome indicates either that the flanking EcoRI sites occur within a modified chromatin structure or that an altered nucleosome arrangement in the vicinity of a flanking EcoRI site is responsible for its location in the nuclease-sensitive internucleosomal (linker) region. Analogous redigestions of the EcoRI-solubilized alpha-oligonucleosomes with either HindIII, MboII or HaeIII (both before and after selective removal of histone H1 by an exchange onto tRNA) produce a self-consistent pattern of restriction site accessibilities. Taken together, these data strongly suggest a preferred nucleosome arrangement within the EcoRI-solubilized subset of alpha-oligonucleosomes, with the centers of most of the nucleosomal cores being approximately 20 bp and approximately 50 bp away from the nearest EcoRI and HindIII sites, respectively, within the 172-bp alpha-DNA repeat. However, as noted above, the clearly preferred pattern of nucleosome arrangement within the EcoRI-solubilized alpha-oligonucleosomes is invariably violated at the ends of every such alpha-oligonucleosomal particle, suggesting at least a partially statistical origin of this apparently non-random nucleosome arrangement.(ABSTRACT TRUNCATED AT 400 WORDS)