Comparative subunit structure of HeLa, yeast, and chicken erythrocyte chromatin.

Abstract

We have compared the chromatin subunit structure of yeast, HeLa, and chicken erythrocyte by analyzing the DNA fragments produced by in situ digestion with staphylococcal nuclease (EC 3.1.4.7) and DNase I (EC 3.1.4.5). The repeat size of the chromatin varies among (and within two of) the three organisms but the size and the structure of the most nuclease-resistant "core" of the repeat is the same. Thus, the interspecies differences in repeat size are due to different lengths of nuclease-sensitive "spacer" DNA between the cores. There also seems to be a difference in the manner of spacing of cores; the transcriptionally active (yeast and HeLa) chromatins have spacings of variable length while the transcriptionally inactive (chicken erythrocyte) has a more regular spacing of cores. Intranuclear digestion of eukaryotic chromatins by staphylococcal nuclease produces a series of discretely sized DNA fragments (oligomers) with sizes that are integral multiples of a basic size (monomer) (1). Evidence indicates that this pattern of fragments results from partial protection of the DNA by a repetitive organization of histones (2,3), which appear in the electron microscope (4, 5) as globular particles [termed nucleosomes (6)], spaced along the chromatin strands.The size of the DNA contained in monomeric and oligomeric nucleoprotein fragments decreases with digestion time; monomers are digested to relatively stable "core particles" containing about 140 base pairs (bp) of DNA and eight histone molecules (7,8). It was suggested for chicken erythrocyte (8) that this behavior reflects the existence of two well defined DNA domains within the repeating unit: a relatively nuclease resistant "core" DNA fragment bound tightly to the eight histones, and a nuclease sensitive "spacer" domain which is distinguished by its relative accessibility to nuclease digestion.To decide whether observed interspecies repeat size differences (1, 7, 9-11) are real differences and whether the core and spacer distinction occurs in genetically active organisms, we have carried out detailed comparative studies of the staphylococcal nuclease digestion of chromatin from three quite different cell types: chicken erythrocytes, HeLa cells, and baker's yeast. These three encompass the range of higher and lower eukaryotes and transcriptionally inactive (erythrocyte) and active (yeast and HeLa) chromatins. We find that the size and structure of the core is similar in all three organisms but the length of the nuclease sensitive DNA between the cores and the mode of spacing varies. MATERIALS AND METHODSPreparation of Nuclei and Nuclease Digestion. Nuclei were isolated and resuspended in digestion buffer as previously described for yeast (10), chicken erythrocyte (8), and HeLa (12) with the following exceptions: for all organisms, 0.1 mM phenylmethylsulfonyl fluoride was added to cell lysis buffer and again during resuspension in digestion buffer; for HeLa cells, lysis buffer contained 1 mM CaCl2, digestion buffer contained 0.1 mM CaC12, and the nuclei were...

Higher order structure in a short repeat length chromatin.

“…2c). This DNA may, however, have been produced by unfolding from the nucleosome (or core particle), a proposal that explains the same phenomenon observed in electron microscope spreads of yeast chromatin (29), which, like neuronal chromatin, also has a very short repeat length (21,22). This latter interpretation is also consistent with the reduced dichroism measurements of depleted neuronal chromatin, which indicated a substantially unwound fiber, even in the presence of divalent cations (Table I).…”

“…However, there is no experimental precedent for H 1 induced higher order folding in a chromatin with a repeat length < 168 base pairs . In yeast chromatin, the absence of an H1 has led to the suggestion that the 30-nm fiber observed (29) with this very short repeat length chromatin (<168 base pairs, (21,22)) may be stabilized by an as yet unidentified molecule (29).…”

Comparison of the DNA repeat length in H1‐ and H5‐containing chromatin (Mature hen erythrocytes, immature chick erythroid cells and hen liver)

“…The size of the repeating unit ranges from 154 base pairs in Aspergillus nidulans [2] to 241 base pairs in the chromatin of sea urchin sperm [7]. As yet the origin of this phenomenon has not been explained but a few possible causes of the observed variability have been excluded [7][8][9]; indeed it has been shown that the rate of cell division, the stage in the cell cycle, the genomic activity, the phosphorylation of H1 and the acetylation of histone Ha and H4 could not be correlated to the variation of the repeat length of chromatin. Noll [1 ] and Morris [2] have suggested that there may be a relationship between the structure of historic H1 (as expressed by the content of basic amino acids) and the length of the DNA repeat in chromatin.…”

“…The variability of the DNA repeat length in chromatin of eukaryotes is now well documented [1][2][3][4][5][6][7][8][9][10][11]. The size of the repeating unit ranges from 154 base pairs in Aspergillus nidulans [2] to 241 base pairs in the chromatin of sea urchin sperm [7].…”

The Primary Structure of Yeast Histone H3

“…In order to confirm that the primary structure differences apply to the entire molecule we have elucidated the completc structure of yeast histone H3. In view of the evidence accumulating which indicates that the packing of DNA in the yeast cell nucleus is closely related to that found in higher organisms [3,4], the existence of major structural changes in the conservative histones could throw a new light on their origin and structurefunction relationships.The genome of yeast is about a hundred times smaller than that of higher vertebrates. This results in a very low histone concentration in the total cell protein.…”