Each level of DNA folding in cells corresponds to a distinct chromatin structure. The basic chromatin units, nucleosomes, are arranged into solenoids which form chromatin loops. To characterize better the loop organization of chromatin we have assumed that the accessibility of DNA inside these structures is lower than on the outside and examined the size distribution of high mol. wt DNA fragments obtained from cells and isolated nuclei after digestion with endogenous nuclease or topoisomerase II. The largest discrete fragments obtained contain 300 kbp of DNA. Their further degradation proceeds through another discrete size step of 50 kbp. This suggests that chromatin loops contain approximately 50 kbp of DNA and that they are grouped into hexameric rosettes at the next higher level of chromatin structure. Based upon these observations a model by which the 30 nm chromatin fibre can be folded up into compact metaphase chromosomes is also described.

The cyclic-AMP concentration in the liver remnant after 70% hepatectomy increases i n a biphasic manner with peak values at 3 and 12 hours, and DNA synthesis begins at 18 hours. Propranolol (dt) injected at 30 minutes after surgery stopped the first wave of cyclic-AMP accumulation, but did not affect the second accumulation or the initiation of DNA synthesis, However, d l , propranolol injected at eight hours equally delayed (by 6 to 8 hours) the second wave of cyclic-AMP accumulation and the initiation of DNA synthesis, Propranolol (cll) did not affect DNA replication per se, since it was totally ineffective after the second wave of cyclic-AMP accumulation had passed and DNA synthesis had been initiated. Propranolol ( d l ) action was not due to a blockade of p-adrenergic receptors, since its d or 1 isomers were separately without effect, as were unrelated P-adrenergic blockers (KO 1313 and M&B 17-803A).On the other hand, an activation of a-adrenergic receptors may be involved in the induction of hepatocyte proliferation, since a-adrenergic antagonists, such as phenoxybenzamine and phentolamine, both delayed and considerably reduced the second wave of cyclic-AMP accumulation and the subsequent initiation of DNA synthesis. It is concluded that the second wave of cyclic-AMP accumulation is somehow associated with the initiation of DNA synthesis.

Calcium, cyclic AMP, and cyclic GMP do not seem to be involved in proliferative activation of postmitotic differentiated cells. Instead, they are intracycle regulators, and we propose the following working model of their control of the initiation of DNA synthesis. While a role for cyclic GMP cannot yet be defined, a brief postmitotic burst of its synthesis might serve to prevent certain activated cells (e.g. 3T3 mouse cells) from being diverted into a nonproliferating (but still activated) G0 state (Figs. 1 and 17). In a latter part of the G1 phase, something happens to stimulate briefly the synthesis of cyclic AMP which, in turn, drives calcium ions from the mitochondria into the cytosol to activate newly synthesized thymidylate synthetase (or other primed enzymic assemblies) (Fig. 1). Having "turned on" their target enzymes, the accumulated cyclic AMP is destroyed and the excess calcium ions are reaccumulated by the mitochondria to avoid interfering with succeeding reactions. This model predicts that persistent changes in cyclic AMP metabolism and the respiration-linked, calcium-accumulating (ion-buffering) activity of mitochondria may be responsible for the sustained growth of tumors.

In a culture medium of pH 7.4 and a folic acid concentration of 100 Lg/liter that contains 5% heatinactivated chicken plasma rather than serum, the rate of proliferation of normal chicken fibroblasts is determined by the concentration of calcium. Proliferation, rapid when the calcium concentration is physiological, decreases when the calcium concentration is reduced. At' a very low calcium concentration, in this culture medium, normal fibroblasts are maintained without proliferation, whereas those infected with Rous sarcoma virus proliferate rapidly. This proliferative inactivity of normal fibroblasts does not involve contact-inhibition, since the effect is observed at low, as well as higher, culture densities. When a physiological amount of calcium is added to cultures of normal fibroblasts that have been maintained in very low calcium-plasma medium for 3 days, labeled thymidine uptake and protein synthesis are strongly stimulated, and cell division follows. The use of heatinactivated chicken serum, instead of plasma, in this medium appears to strongly sensitize normal fibroblasts to the mitogenic action of calcium.In a plasma-containing culture medium of physiological calcium concentration and a folate concentration of 5 ;&g/liter, neither normal nor Rous sarcoma virusinfected fibroblasts proliferate to an appreciable extent. The use of serum, however, instead of plasma results in rapid proliferation of both normal and infected cells, as does increase in the folate concentration of the plasmacontaining medium to 100 ;g/liter.The fact that while normal fibroblasts are maintained without proliferation in low calcium-plasma medium, Rous sarcoma virus-infected fibroblasts proliferate rapidly, indicates that the effect of calcium is regulatory rather than permissive. These results suggest that the proliferation of normal fibroblasts is initiated by a cellular function involving calcium, and that the autonomous proliferation of the neoplastic fibroblasts results either from increased calcium uptake or from an alteration or a bypass of that function. The results also suggest that serum contains a mitogenic factor(s) not present in plasma, possibly a "wound hormone" for fibroblasts.A large amount of evidence has appeared that indicates that calcium and the principal hormones of the calcium homeostatic system are major physiological regulators of the proliferation of thymic lymphoblasts and bone-marrow erythroid cells, and perhaps of liver parenchymal cells and peripheral lymphocytes (1). It has also been shown, by S.D.B., that calcium ion concentration in vitro controls the proliferation of normal chicken fibroblasts in chicken plasma-containing medium, but does not control the proliferation of these cells after they have been infected by the Schmidt-Ruppin strain of Rous sarcoma virus (2, 3). The ability of calcium to regulate normal fibroblast proliferation is obscured in con-675 ventional, but less physiological, serum-containing medium (2, 3). This effect is due to the ability of serum to strongly stimulat...