A preparative method for isolating high-molecular-weight DNA from animal cells is described.This method is based on the use of proteinase K, a powerful proteolytic enzyme with a broad action spectrum, which is very active in the presence of sodium dodecylsulfate and ethylenediamine tetraacetate. The DNA preparation is free of RNA, protein and degrading enzymes.The number-average molecular weight of the native DNA is I90 x lo6, whereas it is 9(& x lo6 for single-stranded DNA, indicating that the DNA molecules do not contain single-stranded nicks.The native DNA molecules range in molecular weight from 40 x lo6 to more than 500 > lo6. Common preparative methods [9] yield mammalian DNA molecules with molecular weights of 5 -20 x lo6, whereasDNA molecules of higher molecular weight (up to 60 x 106) were obtained using a more gentle enzymatic procedure [lo]. Both DNA preparations contain single-stranded nicks caused by the action of nucleases during the time between the death of the organism and the isolation of the DNA. It is obvious that it is impossible to prepare DNA molecules with molecular weights of 1 x loll, due to the well-known degrading effects of mechanical shear during the isolation procedure, but it should be possible to isolate fragments of molecules with molecular weights of several hundred millions containing very few single-stranded nicks, provided the action of degrading enzymes could be totally prevented.

Electron microscopic and biochemical studies demonstrate that the fundamental structure of chromatin depleted of lysine-rich histones is composed of a flexible chain of spherical particles (nucleosomes), about 125 A in diameter, connected by DNA filaments. Such a chromatin preparation can be separated by centrifugation into two fractions which differ in the spacing of the nucleosomes; In one fraction almost all of the DNA is condensed in nucleosomes, while the other fraction contains long stretches of free DNA connecting regions where the nucleosomes are closely packed. The isolated nucleosomes contain about 200 base pairs of DNA and the four histones F2alpha1, F2alpha2, and F2b, and F3 in an overall histone/DNA ratio of 0.97; In such a structure the DNA is compacted slightly more than five times from its extended length; The same basic structure can be visualized in chromatin spilling out of lysed nuclei. However, in this latter case the nucleosomes are very closely packed, suggesting that histone F1 is involved in the superpacking of DNA in chromosomes and nuclei. The chromatin fiber appears to be a self-assembling structure, since the nucleosomal arrangement can be reconstituted in vitro from DNA and the four histones F2alpha1, F2alpha2, F2b and F3 only, irrespective of their cellular origin.

The activation of 5'-nucleotidase in the homogenate as well as in the subcellular fractions, is due to the action of sodium deoxycholate and Triton X-100. Beyond this, both detergents cause a partial solubilization of the enzyme. The solubilized part of the enzyme is not activated during incubation with the detergents. The unsolubilized part of the enzyme is mainly, if not exclusively, activated. The existence of two forms of 5'-nucleotidase has been proposed on the basis of the above statements. These forms probably differ in their stability of binding to the membrane structure.

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