Artificial Structure of Chromatin Derived in the Preparation Process

Abstract

Nuclei were isolated from mouse lymphoma L5178Y cells in the exponential growth phase, and chromatin was prepared by mechanical treatment of the nuclei. The nuclei and the chromatin were then digested to various extents with micrococcal nuclease and the resulting mono- and dinucleosome fractions of the two preparations were compared.
During progressive digestion mononucleosomes from chromatin retained H1 histone and a DNA length of 165 base pairs, whereas those from nuclei released H1 histone and the length of their DNA decreased to 140 base pairs at an early stage of digestion.
These nucleosomal preparations were always associated with nonhistone proteins. The dinucleosomes from nuclei contained larger amounts of nonhistone proteins than those from chromatin, but half of these proteins was released during the process of cleavage into mononucleosomes. The final mononucleosome preparation from nuclei retained 20% less nonhistone proteins than that from chromatin. The contents of nonhistone proteins in mono- and dinucleosomes from chromatin were the same. The electrophoretical distributions of molecular species of nonhistone proteins in mononucleosomes from nuclei and chromatin were different from each other: during digestion the profile of the former changed, whereas that of the latter remained constant.
It is tentatively concluded that both HI histone and nonhistone proteins were bound to nucleosomes more or less loosely in intact nuclei in situ, but that when the nuclear structure was disrupted these proteins became more tightly bound.