Degradation of chromatin in thymus lymphocytes during inhibition of repair of spontaneous DNA damage

Chromatin degradation and cell death were observed after 6-7 h incubation of mouse thymus lymphocytes with 1-beta-D-arabinofuranosylcytosine and hydroxyurea. The time dynamics of both processes was similar. In this case, just as after gamma-irradiation, nucleosomes and their oligomeres were the products of degradation. Puromycin and cycloheximide prevented the toxic action of DNA synthesis inhibitors on thymocytes. It is suggested that the accretion of unrepaired DNA damages to some critical level triggers the process of the internucleosome degradation of chromatin, i.e. implements the program of lymphocyte death.     

Characteristics of the biochemical manifestation of the process of thymic lymphocyte death following UV irradiation

Thymus lymphocyte death caused by UV-radiation is a function of radiation dose. Thymocyte death induced by UV-light, in contrast to gamma-radiation, is not accompanied by chromatin degradation. With UV-irradiation, irreparable DNA breaks are not responsible for thymocyte death. Cycloheximide, but not cysteamine, prevents the UV-induced cell death.     

NAD+-kinase from Neurospora crassa: isolation and properties

The NAD+ kinase (EC 2.7.1.23) of the filamentous fungus N. crassa is localized in cytosol. The activity in the dialyzed cell free extract has a pH optimum 8.3; it utilizes only ATP but not inorganic polyphosphates as a phosphoryl donor. A method for 200-fold purification of NAD+ kinase with a 20% yield has been developed. The procedure includes 105000 g centrifugation, fractionation with (NH4)2SO4, isoelectrofocusing in a Ultrodex layer and preparative electrophoresis in polyacrylamide gel. The molecular heterogeneity of NAD+ kinase was demonstrated by polyacrylamide gradient electrophoresis and by gel filtration through Sephadex G-200. The molecular weights of four individual forms of the enzyme are: 330000-338000, 305000-306000, 215000-229000 and 203000 Da. The Km values for the reaction catalyzed by purified NAD+ kinase for NAD+ and ATP are 3.0 X 10(-4) M and 0.9 X 10(-3) M, respectively.     

Nature of conformational restrictions in nuclear superhelical DNA from thymus lymphocytes

The lymphocyte nucleoids of mouse thymus contain about 40% of rapidly labelled nuclear RNA, about 9% of total intracellular protein and all nuclear DNA. Relaxation of superhelical DNA after thymocyte nucleoids treatment with pronase or RNAase suggests that non-histone proteins and/or RNAs are involved in conformational restrictions in the superhelical domains of cell DNAs. Thymocyte nucleoids proteins are represented by two groups of nonhistone proteins with molecular weights of 50 000-60 000 and 75 000-85 000. An essential role in the appearance of conformational restrictions in thymocyte superhelical DNA belongs to disulfide bonds.