Hydroxylapatite thermal fractionation of chromatin and DNA

Abstract

Chromatin and DNA from developing muscle cultures were fractionated by hydroxylapatite thermal chromatography on the basis of differential thermal stability. A thermal chromatography system was developed in which protein mediated thermal stability of chromatin DNA was maximally expressed. The resulting chromatin and DNA elution profiles were similar to thermal denaturation profiles in low ionic strength solution. Additional studies showed this system was able to detect protein stabilization of DNA in in vitro nucleohistone preparations. Although some protein remained bound to hydroxylapatite during chromatin thermal elution, it did not affect the denaturation or elution behavior of free DNA on the same column. These studies show that fragments of chromatin or DNA can be segregated on the basis of differential thermal stability by hydroxylapatite chromatography.

Deoxyribonucleic acid-protein and deoxyribonucleic acid interstrand crosslinks induced in isolated chromatin by hydrogen peroxide and ferrous ethylenediaminetetraacetate chelates

“…The sheared, clarified chromatin exhibited the following spectral ratios: A260/Am = 0.97; A2(a¡A2W = 1.68; maximum/minimum = 1.36; A260/A220 = 42. DNA was purified from sheared chromatin as described by Saffitz & Caplan (1978) and dialyzed against 1 mM NaC104. This purified DNA has an i020w of 12.96, which corresponds to a molecular weight of 2 X 106.…”

Attempted separation of transcriptively active and inactive chromatin by hydroxylapatite thermal chromatography

“…Methods for the preparation of developing muscle cultures, the labeling of chromatin DNA in culture, and the preparation of chromatin from in vitro and in vivo muscle tissue are provided in the preceding communication (Saffitz & Caplan, 1978). Muscle culture chromatin and D N A were fractionated on hydroxylapatite after shearing chromatin at either 15 000 or 30 000 psi' in a French pressure cell (Aminco).…”

“…and ethanol precipitated. The purified D N A was sheared at 30 000 psi in a French pressure cell to yield fragments of approximately 300 base pairs (Ordahl et a\., 1976;Saffitz & Caplan, 1978). Sheared D N A was excluded from Sephadex G-100 equilibrated with NTE buffer at room temperature and then passed over Chelex-100 in N T E buffer.…”

“…mized (Saffitz & Caplan, 1978). In this report, we studied the distributions of transcribed vs. nontranscribed sequences, and repetitive vs. nonrepetitive sequences within fractions of chromatin and DNA separated by hydroxylapatite thermal chromatography.…”

“…Insight into the regulation of differential gene expression could be provided were one able to study the structural and functional features which distinguish transcriptively active and inactive chromatin. Toward this end, several methods for fractionating chromatin have been developed (for a recent review, see Gottesfeld, 1977; for a list of chromatin fractionation references, see Saffitz & Caplan, 1978).We have studied one such method developed by McConaughy & McCarthy (1972) in which chromatin is fractionated by hydroxylapatite chromatography on the basis of differential thermal stability. A relationship between chromatin thermal stability and template activity is suggested from comparisons of chromatin and DNA denaturation in low ionic strength solution (Huang & Bonner, 1962;Marushige & Ozaki, 1967;Paoletti & Huang, 1969;Li & Bonner, 1971) and from observations that condensed chromatin denatures at higher mean temperatures than extended chromatin (Frenster, 1965;Duerksen & McCarthy, 1971).…”