Abstract
Chromatin solubility was observed at several concentrations of various cations. Spermine and spermidine precipitated (50%) chromatin at about 0.2 mM, Ca2+ and Mg2+ at about 1-2 mM, and Na+ at about 100 mM. Further increases in cation concentration induced more aggregation, but eventually excess cation increased chromatin solubility so that 50% solubility was observed again at 60 mM Mg2+ and 180 mM Na+. H1 histone was 50% released by 80 mM MgCl2 or 425 mM NaCl. Combinations of MgCl2 and NaCl showed that Mg2+ and Na+ are synergistic in the induction of aggregation in lower concentrations (less than 2 mM) of Mg2+ but antagonistic at higher concentrations, and a similar effect of NaCl on spermidine-induced precipitation was shown below and above about 0.2 mM spermidine. At 5 mM, MgCl2 proved capable of precipitating chromatin depleted of H1 histone, but no concentration of NaCl was capable of doing so. These phenomena can be rationalized by supposing that neutralization of chromatin by any cation (including H1 histone) favors aggregation and also that cross-linking of chromatin fibers by multivalent cations (including H1 histone) is also critically important. The exchange of H1 histone between chromatin fragments was tested in various concentrations of different salts. H1 exchange was correlated with chromatin aggregation rather than with ionic strength and thus appears to depend on fiber to fiber contact. Under conditions where H1 exchanges between chromatin fibers that are permitted to make contact with each other, no H1 exchange occurred between chromatin inside the nucleus and chromatin outside, even though H1 histone is capable of passage through the nuclear membrane.
Highlights
5 mM, MgCl, proved capableof precipitating chromatin MgC1,induced precipitation to NaCl to40 mM
The notion that nonuniform distribution of H1 from one fibers that are permitted to make contact with each region to another in chromatin could have physiological sigother, no H1 exchange occurred between chromatin nificance [8, 12,13,14,15,16,17,18] would be nonsense if H1 histone were inside the nucleus and chromatin outside, even thougfhree to wander over the surfaceof chromatin without restric
A 0.2-ml aliquot of the supernatantwas diluted into 2 ml of solution by adding 2% SDS, and the absorbance a t 260 nm was measured the case of MgClz,thethresholdforaggregationoccurred about where chromatin shows apparent saturation by Mg2+ [29], it may not be fully neutralized at this point [30]
Summary
Preparatwn of Nuclei-All steps were carried out at 0-4 "C. Cell cultureand radioactive labeling of H1 histone were as described previously [19]. Preparation of Chromatin-Nuclei were washed two to three times with buffer A (0.3 M sucrose, 50 mM Tris, 25 mM KCl, 5 mMMgC12, pH 6.5) before digestion. Nuclei were pelleted (5 min a t 5,000 X g), lysed by ml of 'H-labeled HeLa nuclei suspension (AzfiD= 26) in buffer A was pelleted and mixed with 1 mi of the above chromatin solution and incubated at 4 "C for the desired time. Protein Analysis and Determination of Radioactivity-Chromatin samples were analyzed by 12.5% SDS-polyacrylamide gel electrophoresis, essentially according to Laemmli [27]. The DNA that was precipitated overnight in 2.5 volumes of ethanol a t -20 "C with centrifuged at 8000 X g for 10 min and redissolved in electrophoresis sample buffer.
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