Abstract
Sedimentation analysis has been used to compare the structure of 30-nm chromatin fibers, isolated and digested under conditions that maintain the native structure, with relaxed-refolded chromatin. The native chromatin fibers show sharp, ionic strength-dependent changes in sedimentation coefficient that are not apparent in relaxed-refolded fibers. The first transition at approximately 20 mM ionic strength reflects the organization of the 10-nm polynucleosome chain into a loose helically coiled 30-nm fiber. Between 20 and 60 mM ionic strength there is considerable interaction between nucleosomes within the coils to generate a stable helical array with 12 nucleosomes/turn. Above 60 mM ionic strength the helical coil continues to condense until it precipitates at ionic strengths slightly greater than those considered physiological, indicating that there is no end point in fiber formation. The data is incompatible with a solenoid model with 6 nucleosomes/turn and also rules out the existence of a beaded subunit structure.
Highlights
From the Cellular Oncology Group, Division of Biological Sciences,National Research Council of Canada, Ottawa, Ontario KIA OR6, C a d a Sedimentationanalysis has been used to compare the Sedimentation analysis is one of the few solution techniques structure of 30-nm chromatin fibers, isolated and di- that permits chromatin to be studied over a wide range of gested under conditions that maintain nthaetive struc- ionic strengths and has been used to provide data that is ture, with relaxed-refolded chromatinT. he native considered to support each of the above models [2,3,4,5, 8,9,10,11]
For small oligomers the s ~val~ues, ar~e determined for each oligomer directly rather than using the mass average of a previously fractionated sample [11].In our hands, mass averages could never be accurately determined for mixtures of small oligomers
Quite clearly, refolded oligomers do not have the same sedimentationcharacteristics as native fibers
Summary
From the Cellular Oncology Group, Division of Biological Sciences,National Research Council of Canada, Ottawa, Ontario KIA OR6, C a d a Sedimentationanalysis has been used to compare the Sedimentation analysis is one of the few solution techniques structure of 30-nm chromatin fibers, isolated and di- that permits chromatin to be studied over a wide range of gested under conditions that maintain nthaetive struc- ionic strengths and has been used to provide data that is ture, with relaxed-refolded chromatinT. he native considered to support each of the above models [2,3,4,5, 8,9,10,11]. From the Cellular Oncology Group, Division of Biological Sciences,National Research Council of Canada, Ottawa, Ontario KIA OR6, C a d a Sedimentationanalysis has been used to compare the Sedimentation analysis is one of the few solution techniques structure of 30-nm chromatin fibers, isolated and di- that permits chromatin to be studied over a wide range of gested under conditions that maintain nthaetive struc- ionic strengths and has been used to provide data that is ture, with relaxed-refolded chromatinT. Chromatin fibers show sharp, ionic strength-dependentIn thispaper we have used this technique to study the changes changes in sedimentation coefficient that are not ap- of both native and relaxed-refolded liver chromatin over the parent in relaxed-refolded fibers. Is not consistent with either the solenoid or the superbead models for the structureof the 30-nm fiber, but is compatible with a helical coil arrangement containing 12 nucleosomes/ turn. 60 mM ionic strength the helical coil continues to condense until it precipitates at ionic strengths slightly
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