[Isolation of Ca2+, Mg2+-dependent nuclease from calf thymus chromatin]. / Vydelenie Ca2+, Mg2+-zavisimoi nukleazy iz khromatina timusa telenka.

Ca2+,Mg2+-dependent nuclease was isolated from calf thymus chromatin by stepwise chromatography on DEAE-Sepharose, CM-Sephadex and DNA-Sepharose. The enzyme was purified more than 700-fold. SDS-PAGE electrophoresis revealed one protein band possessing an enzymatic activity. The molecular mass of the nuclease as determined by gel filtration is 25700 Da, that determined by 12% SDS polyacrylamide gel electrophoresis is 28,000 Da. In the presence of various ions the enzyme activity decreases in the following order: (Ca2+ + Mn2+) greater than (Ca2+ + Mg2+) greater than Mn2+; the pH optimum is at 8.0. In media with Mg2+, Ca2+, Co2+ and Zn2+ the nuclease is inactive. Some other properties of the enzyme are described.

[Immunochemical analysis of changes in the structure of the nuclear matrix in thymocytes of irradiated rats]. / Immunokhimicheskii analiz izmenenii struktury iadernogo matriksa timotsitakh obluchennykh krys.

It was shown that reactivity of the nuclear matrix of thymocytes for antibodies against chromatin of the control and irradiated thymocytes and PDN did not change immediately and increased markedly 2 h following irradiation of rats with a dose of 10 Gy. The method of immunoblotting failed to reveal any qualitative differences in the protein content of the thymocyte nuclear matrix of the control and exposed rats.

[Postirradiation changes in the chromatin structure of thymocytes in irradiated rats]. / Postradiatsionnye izmeneniia struktury khromatina timotsitov obluchennykh krys.

In this work the antibodies were obtained against chromatin isolated from thymocytes of intact and irradiated rats (2 h after exposing to 10 Gy) and against polydeoxyribonucleotides (PDN) extracted from thymus nuclei 6 h following irradiation. All the antibodies under study reacted more readily with the chromatin obtained from the thymus of exposed rats than with the control chromatin. The complexes of DNA with the most firmly bound non-histone proteins, obtained from the three objects under study, reacted with the antibodies with equal efficiency. Thus, a higher reactivity of PDN and chromatin from thymocytes of exposed rats was associated with the decondensation of the latter leading to an increase in availability of a part of antigenic determinants. Using the immunoblotting method we failed to discover any qualitative differences in the protein composition of the chromatin from control and exposed rats.

[Immunogenic, tissue-specific, antigenic determinants of thymocyte chromatin--firmly bound to nonhistone proteins]. / Immunogennye tkanespetsificheskie antigennye determinanty khromatina timotsitov--prochno sviazannye negistonovye belki.

Rat thymocyte chromatin was dissolved in 1.0 or 2.0 M NaCl and the dissociated proteins were separated by ultracentrifugation or gel filtration on Sephadex G-200. Rabbit antibodies against tissue-specific antigenic determinants of thymus chromatin interact with "depleted" deoxynucleoproteins and not with dissociating proteins. The DNA complex with firmly bound proteins obtained in 2 M NaCl was treated with DNAase I; this treatment had no effect on proteins interaction with antibodies. Thus, tissue-specific antigenic determinants can be related to the most firmly bound nonhistone proteins.

Chromatin regions, released by endogenous nucleases, are enriched in immunogenic tissue-specific proteins.

Localization of immunogenic tissue-specific proteins in chromatin regions, hypersensitive to endogenous nucleases, has been studied using rabbit antibodies against rat thymus chromatin. It is shown that the first 1-2,5% of the chromatin (calculating on DNA), released by Mg2+-, Mn2+- and Ca2+/Mg2+-dependent nuclear endonucleases are drastically enriched in tissue-specific antigenic determinants. The released chromatin fractions are found to contain a heterogeneous set of nonhistone proteins and are deficient in histones. The cleavage of nuclear DNA by endogenous acidic nuclease, independent on bivalent ions, resulted in a significantly less enrichment of the released fractions with immunogenic proteins.

[Mechanism of chromatin degradation in thymocytes of irradiated rats. 6. Postradiation changes in the activity of poly(ADP-riboso)-polymerase]. / Mekhanizm degradatsii khromatina v timotsitakh obluchennykh krys. Soobshchenie 6. Postradiatsionnye izmeneniia aktivnosti poli(ADF-ribozo)-polimerazy.

A biphase change in poly (ADP-ribose) polymerase activity of the thymocyte chromatin was observed after 10 Gy irradiation of rats: during the first minutes the incorporation of 14C-NAD increased by 40% then started decreasing to make 110, 60 and 35% after 1, 2 and 3 h, respectively. Irradiation of rat thymus chromatin in vitro sharply decreased poly (ADP-ribose) polymerase activity. The possible role of changes in the poly (ADP-ribose) synthesis in the activation of nuclear Ca/Mg-dependent endonuclease and in the postirradiation degradation of the thymocyte chromatin is discussed.

Comparison of some properties of chromatin non-histone proteins and nuclear sap proteins.

The properties of rat liver and thymus non-histone and nuclear sap proteins were compared. The distribution of total, labile-bound and 0.35 M NaCl extractable non-histone proteins from one organ on polyacrylamide-gel electrophoresis in the presence of sodium dodecylsulphate is quite similar. On electrophoresis non-labelled and 32P-labelled non-histone and nuclear sap proteins from one organ differ from one another both qualitatively and quantitatively. We did not find an appreciable difference between non-labelled non-histone proteins isolated from liver and thymus. The distribution of 32P-labelled non-histone proteins from various organs differs quantitatively rather than qualitatively. Non-labelled and 32P-labelled nuclear sap proteins from liver and thymus differ significantly. 'Free' nuclear sap proteins and the proteins of ribonucleoprotein particles from thymus nuclei contain a great quantity of identical polypeptides, whereas other polypeptides are specific to each of these protein fractions. Upon incubation of nuclei with [gamma-32P]ATP the label is incorporated into all the fractions of nuclear protein. The nuclear proteins are phosphorylated at decreasing rates in the order: labile-bound non-histone proteins greater than firmly bound non-histone proteins greater than 'free' nuclear sap proteins = proteins of ribonucleoprotein particles greater than histones. Nuclear sap and non-histone proteins contain protein kinases capable of phosphorylating both these proteins and histones. Histone phosphorylation is sharply inhibited after addition of DNA, the protein kinases of nuclear sap phosphorylating less effectively the histones complexed with DNA than the non-histone proteins. Both non-histone and nuclear sap proteins contain fractions interacting in vitro with DNA. Denatured DNA binds twice as much 32P-labelled nuclear sap proteins and a little more 32P-labelled non-histone proteins than native DNA. Denatured DNA binds non-histone and nuclear sap proteins much more effectively than native DNA. It was shown by the membrane filter technique that the major part of the nuclear sap and non-histone proteins interacting with native DNA binds to it non-specifically. A certain portion of non-histone and nuclear sap proteins interacts specifically with homologous denatured DNA. The possible role of non-histone and nuclear sap proteins in the regulation of transcription is discussed.