Structures retaining many of the morphological features of nuclei may be released by lysing HeLa cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids contain few chromatin proteins. We have shown that the DNA of nucleoids is quasicircular and supercoiled by measuring spectrofluorometrically the amount of the intercalating dye, ethidium, bound to unirradiated and y-irradiated nucleoids. Ethidium binds to nucleoids in the manner characteristic of the binding to superhelical DNA: at low concentrations more ethidium binds to unirradiated nucleoids than to their y-irradiated counterparts with broken DNA, and at higher concentrations less ethidium binds to the unirradiated nucleoids. The quasi-circles in nucleoids are 22 times less sensitive to y-irradiation than are circles of pure PM2 DNA: they must contain about 2.2 x lo5 base pairs.The constraints that maintain the quasi-circularity of nucleoid DNA are very resistant to extremes of temperature and alkali; some remain under conditions in which the duplex is denatured. The constraints are destabilised by ethidium suggesting that they are stabilised by free energy of supercoiling. Proteolytic enzymes, but not ribonucleases, remove the constraints. Possible structures for the constraining mechanism are discussed. Circular duplexes of DNA in which both strands of the duplex are covalently continuous possess one remarkable property not shared by their counterparts with broken strands: the two strands in the intact molecule are interlocked and can only be completely separated by severing one of the strands i.e. by breaking phosphodiester bonds (see [1 -31 for a fuller discussion). We have shown that the DNA of higher cells is subject to the same kind of topological constraint as that found in circular molecules of DNA. Our approach was to lyse cells in solutions containing non-ionic detergents and high concentrations of salt to release structures that retain many of the morphological features of nuclei. These nucleoids contain few of the proteins characteristic of chromatin and sediment in sucrose gradients containing ethidium in the manner characteristic of superhelical DNA. Breaking the DNA by y-irradiation abolished the characteristic behaviour [4-81 (see also [9-111). We concluded that nucleoid DNA was made quasi-circular by
Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction endonuclease and the cages - and any associated DNA - sedimented free from unattached DNA. The cage-associated DNA is purified and completely fragmented using the same restriction endonuclease. Equal weights of fragmented DNA are separated by gel electrophoresis, transferred to a filter and the relative amounts of the alpha, beta and gamma globin genes on the filter determined by hybridisation to the appropriate probes. The alpha genes, unlike the beta and gamma genes, resist detachment from the cage and so must lie close to the point of attachment to the cage. Our ability to map these genes implies that sequences cannot be attached at random to the cage; rather, specific sequences must be attached, so looping the DNA.
SUMMARY Parathyroid hormone has a direct effect on the osteoclast population present at the time of administration of the hormone. There was a significant stimulation of nuclear RNA synthesis at the earliest time (1½ hr.) at which the measurement could be made. This is followed by increased production of cytoplasmic RNA which reaches its maximum after a considerable time-lag (7 to 12 hr. after parathyroid extract (PTE)). The increase in cytoplasmic RNA is accompanied by a corresponding stimulation of protein and mucoprotein synthesis in the osteoclasts and the effect persists at least until 24 hr. This time-lag and the relatively long duration of the effect on protein synthesis can be correlated approximately with the effect on the plasma calcium level. It is suggested therefore that the rise in plasma calcium is mainly due to the increased cellular activity of the osteoclasts and the resulting increased bone resorption. The opposite effect on the osteoblast system has about the same time-sequence and would complement the effect on the osteoclast system. At about the same time as the maximum increase in cellular activity in the osteoclasts is observed, a significant effect on RNA synthesis in the endosteal mesenchymal cells, the precursors of the osteoclasts, becomes apparent. This is closely followed by a rise in the number of osteoclasts which is first apparent at 17 hr. after PTE and is maximal by 24 hr. Consequently, the rise in the number of osteoclasts is a secondary effect and is not responsible for the initial rise in plasma calcium which occurred much earlier. It is suggested that the increase in osteoclast numbers follows as a result of the increased metabolic activity of the osteoclast population present when the hormone was injected. There is a depression of RNA synthesis in both the osteoblasts and their precursors, the preosteoblasts. This means that the hormone has opposite effects, not only on the osteoblasts and osteoclasts, but also on their respective precursors, indicating that osteoprogenitor cells contain a mixture of cells with two main lines, those differentiating in an osteoblastic or osteoclastic direction, respectively.
Structures resembling nuclei may be released by gently lysing human cells in solutions containing non-ionic detergents and high concentrations of salt. These structures, which we call nucleoids, sediment in sucrose gradients containing the intercalating agent, actinomycin D, in the manner characteristic of superhelical DNA. We have determined the concentration of actinomycin that minimises the rate of sedimentation of nucleoids. At this concentration, we have determined the amount of drug bound per base pair of DNA by means of a double-labelling procedure. Assuming that each molecule of actinomycin bound to nucleoid DNA unwinds the double helix by 26", we calculate that there is one supercoil every 90-180 base pairs in nucleoid DNA. These values lie within the range found for the circular DNA molecules of plasmids and viruses.Recent evidence suggests that chromatin is composed of subunits: sections of DNA about 200 base pairs in length are condensed by histones into roughly spherical particles to yield a flexible chain of repeating subunits [I -31. It is not yet clear how the DNA is folded within the repeating unit. The three-dimensional structure of circular molecules of pure DNA has been described in some detail [4]. The description is based upon a distinction between the right-handed helical turns of the double helix and any superhelical turns that might be superimposed upon the duplex turns. The superhelical turns are lost spontaneously when phospho-diester bonds are broken in either of the backbone strands of the double helix. We have recently shown that the DNA of higher cells is supercoiled, like the circular DNA of viruses [5,6]. We have now determined the degree of supercoiling in the nuclear DNA of human cells.When intercalating agents (e.g. ethidium bromide, actinomycin D) bind to an intact and circular molecule of DNA, the right-handed double helix is unwound and the number of superhelical turns is altered. The conformational changes associated with intercalation are reflected by alterations in the sedimentation coefficient of DNA. Intact circles of supercoiled DNA are compact and sediment more rapidly than their broken counterparts which have lost supercoils and so have become extended. The sedimentation coefficient of broken or nicked DNA changes little in the presence of increasing concentrations of intercalating agent, whereas that of supercoiled DNA changes dramatically. Initially intercalation removes super-
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