Differential dissociation of chromatin digests: a novel approach revealing a hierarchy of DNA-protein interactions within chromatin domains.

We describe here a novel approach to the dissection of chromatin structure by extracting DNA fragments from digested nuclei irreversibly immobilized (via proteins) on Celite columns. Three successive gradients (NaCl, LiCl-urea, temperature) are used to release three families of DNA fragments: namely, the 'DNA adherence' classes DNA-0, DNA-I and DNA-II, respectively. This 'protein image' DNA chromatography separates DNA fragments in accordance with the tightness of their bonds with proteins in situ. There are at least two DNA-skeleton attachment sites differing from each other by their resistance to the dissociating agents used as well as their susceptibility to DNAase I and S1 nuclease treatments, DNA cross-linking and single-stranded breaks. Several lines of evidence show a specific, topological rather than chemical, DNA-protein linkage at the tight attachment site. A hierarchy of chromatin loops demarcated by these attachment sites was determined. The technique described is generally applicable and can be used both to probe DNA-protein interactions and to map specific DNA sequences within the chromatin domain.

Ribosomal and informational ribonucleoprotein complexes of animal cells. Study on rat liver ribonucleic acids as constituents of ribonucleoprotein complexes by chromatography on nucleoprotein-celite columns.

A novel method of RNA fractionation has been developed. Nuclear and cytoplasmic rat liver RNA species were fractionated as constituents of corresponding ribonucleoprotein particles, which were previously adsorbed on a Celite-column by their protein component. The fractionation is based on a dissociation of the particles (linear concentration gradient of LiCl and urea with subsequent temperature gradient), which results in a gradual release of the RNA molecules from ribonucleoprotein complexes. Thus the fractionation is in accordance with the tightness of the RNA-protein bonds. A gradient elution of RNA from a nucleoprotein-Celite column permitted fractionation of both ribosomal and rapidly labelled non-ribosomal RNA. The latter, both nuclear and cytoplasmic, could be separated by chromatography on nucleoprotein-Celite columns into two main fractions (components I and V). In cytoplasmic RNA components I and V presumably correspond to mlRNA (messenger-like RNA of free cytoplasmic particles) and mRNA (template RNA associated with ribosomes) respectively.