Structure and properties of rat-thymus deoxyribonucleoprotein. A comparison of the electric birefringence and other properties of deoxyribonucleoprotein prepared by various methods

1. A comparison has been made of the properties of dNP (deoxyribonucleoprotein) prepared in various ways from rat thymus. The variables studied were: (a) extent of homogenization; (b) concentration of various metal ions; (c) concentration of EDTA; (d) extent of dialysis; (e) concentration of dNP. The chief method used for studying the dNP was that of electric birefringence; this gives information about the optical and electrical properties of molecules oriented in an electric field and about their length. 2. The extent of homogenization during the preparation affects the birefringence properties only slightly and the solubility properties of the constituent DNA not at all. However, the protein content of the dNP decreases after repeated homogenization. 3. In the presence of added Ca(2+), Mg(2+) or Na(+) an insoluble product is obtained. 4. The presence of added EDTA affects the birefringence properties only slightly. 5. In the final stage of preparation, both very limited dialysis and extensive dialysis (in terms of volume ratio) cause partial insolubility of the final product. 6. dNP prepared at low concentrations resembles in some respects dNP prepared at higher concentrations and then subjected to limited enzyme action but it is not degraded (as by extensive enzyme action); ultracentrifuge studies of the constituent DNA confirm that no degradation of the DNA has occurred. 7. It is concluded that dNP prepared at low concentrations or by extensive dialysis is denatured and that preparation of ;native' material is favoured by maintenance of a high dNP concentration and by fairly limited dialysis.     

Ultracentrifuge studies of histone fractions from calf thymus deoxyribonucleoprotein

Molecular weights and sedimentation coefficients of four major fractions of calf thymus histones were measured. The minimum molecular weights were determined in concentrated solutions of guanidine hydrochloride. The results indicate that, with the possible exception of fraction F3, the fractions are heterogeneous. Comparisons in 0.1m-sodium chloride suggest that fraction F1 does not aggregate and show that fractions F2(a) and F3 aggregate to form larger complexes than does fraction F2(b). The degree of aggregation of each fraction is independent of pH in the range pH1-7. Detailed studies with fraction F2(b) have confirmed that the change in sedimentation coefficient observed as the sodium chloride concentration of the solution is increased results from increases in the apparent molecular weight of the sedimenting units. It has been found that the molecules of fraction F2(b) are present as single molecules only in sodium chloride solutions of 33mm or less. At these low concentrations the effects of charge greatly increase the concentration dependence of the sedimentation rate; the results can, however, be interpreted by using the theory developed by Alexandrowicz & Daniel (1963) and Daniel & Alexandrowicz (1963).     

Effects of irradiation on RNA synthesis primed by calf thymus deoxyribonucleoprotein treated with salt and salt-urea.

1. The effects of ionizing radiation on the activity of calf thymus templates were examined in a Escherichia coli RNA polymerase system. 2. The template activity of native and 2 M NaCl-5M urea-treated deoxyribonucleoproteins was enhanced by relatively low doses of irradiation, while that of 2 M NaCl-treated deoxyribonucleoprotein was not enhanced by irradiation. 3. The template activity of purified DNA was markedly decreased by irradiation, while that of native deoxyribonucleoprotein, 2 M NaCl-treated, and 2 M NaCl-5 M urea-treated ones were slightly decreased at a higher dose range. The doses for 50% inactivation of these templates were 1.3, 210, 140, and approximately 200 krad, respectively.     

Structure and repair of rat thymus DNA during long-term irradiation

During long-term fractionated irradiation (0.5 Gy, daily) the molecular weight of single-stranded DNA of the thymus of exposed rats remained the same as that of intact animals till the dose of 25 Gy had been cumulated. The integrity of the DNA structure was ensured by the repair of DNA and elimination of cells with unrepaired lesions. The role of repair decreased and the elimination of cells increased with increasing cumulative dose.     

Solubility properties of alpha-reduced paramyosin.

It is now believed that the reduced form of alpha-paramyosin is that found in living adductor muscles of molluscs. We have studied the solubility of a preparation of alpha-paramyosin obtained under reducing conditions. In contrast to the solubility profile of beta-paramyosin, the alpha-paramyosin, the alpha-preparation showed a rapid, almost linear decrease in solubility over the ionic strength range 0.35-0.25 at neutral pH. Solubility in this range was further decreased by the presence of physiologically small amounts of calcium ion. Lactate ion, which can accumulate during anaerobic glycolysis in molluscan muscles, also decreases the solubility at a level of 50 mM. In addition, the type of paracrystal formed by alpha-paramyosin differs greatly from those of beta-paramyosin and paracrystal formed in the presence of lactate differs from those formed in buffer solutions. Reduced alpha-paramyosin is more sensitive to the above parameters than the preparations made without reducing agents. Moreover, the pH and ionic strength ranges in which greatest change in solubility behaviour occurs are physiologic, as are the calcium and lactate ion levels effective in increasing intermolecular interactions. A model is proposed for alpha-paramyosin in which the extra 5% presumably removed in beta preparations is a "sticky head" which protrudes from one end of the molecule and confers on it an increased tendency for interaction, particularly at physiological ionic strength. Such molecules would be capable of promoting interactions between thick filaments which contain them, providing a means of accounting for the pH dependent stiffness observed in glycerinated preparations of molluscan catch muscles.     

Phosphorylation of rat thymus histones, its control and the effects thereon of gamma-irradiation.

The phosphate content of rat thymus histones was determined. As expected for a replicating tissue, histones 1 and 2B were more phosphorylated and had higher 32P uptakes than did histones from resting liver nuclei; the other histones all showed 32P uptake, but the phosphate content and uptake of histone 2A was about half that for liver histone 2A. When thymus nuclei were incubated in a slightly hypo-osmotic medium, non-histone proteins and phosphorylated histones were released into solution; this was enhanced if ATP was present in the medium. [gamma-32P]ATP was incorporated into non-histone proteins, including protein P1, and into the ADP-ribosylated form of histone 1; negligible 32P was incprporated into the other, bound, histones. Histones 1 and 2B added to the incubation medium were extensively, and histones 2A and 4 slightly, phosphorylated. Histones released by increasing the ionic strength of the medium were phosphorylated. Added lysozyme and cytochrome c were neither bound nor phosphorylated, but added non-histone protein P1 was phosphorylated, causing other histones to be released from the nuclei, especially histones 2A and 3. The released histones were phosphorylated. gamma-Irradiation decreased 32P uptake into the non-ADP-ribosylated histones 1 and 4; phosphorylation of histone 1 in vitro was unaffected. The importance of non-histone proteins, ATP availability and nuclear protein kinases to the control of histone phosphorylation in vivo is discussed.