Study of heterogeneity of chromatin endonuclease fragments, using fractionation by step-wise endonucleolysis

Isolated nuclei from rat liver were incubated at different time intervals under conditions, optimal for manifestation of the Ca, Mg-dependent endonuclease activity. After each of the 6 endonucleolyses chromatin was extracted and 6 chromatin fractions (I--VI) and "residual" chromatin were collected. A comparative analysis of the "early" (I--III) and "late" chromatin fractions revealed an increased RNA content in the "late" fractions and changes in the composition of the non-histone proteins. Electrophoresis in acrylamide gel concentration gradient demonstrated that fractions I--III predominantly contain high molecular weight fragments whereas fractions IV-VI are represented by highly fragmented chromatin. Each fraction was characterized by peculiar shapes of alkaline denaturation curves and by heterogeneity of charges of their constituent chromatin fragments.     

Effect of the conditions of isolation and incubation of the nuclei on digestion of DNA chromatin by calcium- and magnesium-dependent endonuclease. Change in the spectrum of chromosomal proteins and possible role of DNA-protein interactions]

Digesting of chromosomal DNA of interphase rat liver nuclei by Ca, Mg-dependent endonuclease in situ in the presence of chelating agents results in the appearance of the soluble DNP--up to 30% of the total DNA. In addition, 50% of the chromatin is solubilised after mild ultrasonication. In the absence of the chelating agents the degree of fragmentation is considerably increased. The process is accompanied by a loss of some histone and nonhistone chromosomal proteins; the nonhistone proteins are lost selectively. The preliminary removal of the nuclear membrane and significant part of the proteins by tritone X-100 promotes the chromatin degradation and the appearance of low molecular weight fragments. The DNA-fragments of solubilised chromatin are similar to the DNA-fragments of residual chromatin, but in the presence of the chelating agents the latter does not contain monomeric fragments.     

Structural organization of the bacterial nucleoid using endonucleolysis

The fragmentation of bacterial deoxyribonucleoprotein (bDNP) in the spheroplasts of Escherichia coli, Serratia marcescens, Pseudomonas fluorescens and Micrococcus luteus by bacterial intracellular Ca2+ or Ca2+, Mg2+-dependent endonucleases in situ was studied. An electrophoresis of the extracted nuclease-split bDNP revealed the presence of high molecular weight (nuclease-resistant) and low molecular weight multiple fragments (100--120 nucleotide pairs). The electrophoretic mobility of the smallest nuclease-split DNA fragments in all bacterial species under study was similar, indicating the orderly structure of bDNP. Two total fractions whose electrophoretic mobility corresponded to that of the histones H2a and H2b from calf thymus were prevalent in the spectrum of acid-soluble bDNP proteins of gram-negative species. The heterogeneity of DNP with respect to its sensitivity to nucleases, is interaction with membranes and protein distribution pattern were revealed by treatment of the bacterial nucleoid with endogenous endonucleases, which probably reflects differences in the functional state of individual sites of the genome.     

Non-histone proteins of soluble nucleoproteins released from mouse myeloma nuclei by mild micrococcal nuclease digestion

Mono- and dinucleosomes preferentially cleaved from mouse myeloma chromatin by very mild micrococcal nuclease digestion at 0 degree C are soluble and are released from nuclei under near-physiological conditions in which normal nucleosomes containing Hl are insoluble. These nucleosomes are highly enriched in RNA, high-mobility-group proteins and a unique subset of other non-histone proteins. They are nearly devoid of histone Hl and contain DNA significantly less methylated than whole myeloma DNA, indicating that they comprise a subset of genomic sequences. Previously we have shown that this fraction is enriched in transcribed DNA sequences. Non-histone proteins that co-sedimented with readily solubilized nucleosomes included many of the most basic, low-to-moderate molecular weight chromosomal proteins. Many of these proteins were also preferentially acetylated in vivo. The residual, pelleted chromatin was highly enriched in high molecular weight proteins (greater than 60 000), and very depleted in medium molecular weight proteins. Readily solubilized nucleoproteins sedimenting like mononucleosomes were partly resolved by electrophoresis, under non-denaturing conditions, into several subfractions differing significantly in non-histone protein contents. Methods described here should be useful for identifying and isolating non-histone proteins bound to nucleosomes and other chromatin regions that are structurally and functionally unique.     

Vitellogenin-specific Non-histone Chromatin Protein

A non-histone chromatin protein with a molecular weight of about 10,000 daltons was purified from the liver of egg-laying hens. The protein binds tightly to hen genomic DNA fragments carrying at least a part of the vitellogenin structural gene. This vitellogenin-specific non-histone chromatin protein is induced by estrogen before, or in parallel with vitellogenin synthesis in the liver of male chickens, in which vitellogenin is not usually synthesized.     

The possible role of endogenous nucleases in the structural organization of chromatin]

Two fractions of rat liver nuclei with different buoyant density have been obtained. The electrophoretic analysis of the oligonucleosome patterns of DNA out of nuclei of these two fractions revealed different levels of activity in endonucleases. In case of inhibition during the extraction of activity in Ca, Mg-dependent endonucleases, the average size of high polymeric DNA is larger for nuclei with bigger buoyant density (fraction I) than for nuclei with smaller ones (fraction II). This finding is evidence of in situ existence of two pools of liver nuclei with different endogenic nuclease activities. In nuclear chromatin fraction I DNA is torsionally stressed; in fraction II it is relaxed that correlates with larger activity of endonucleases and smaller buoyant density of this fraction. A hypothesis on a possible role of endonucleases in chromatin structure organization has been put forward. According to this hypothesis a modulation of activity in nuclear endonucleases can determine different packaging and activity of chromatin from different pools of cellular nuclei.     

Isolation of Ca/Mg-dependent endonuclease of cell nuclei as the basic component in the chromatin nuclease complex of human lymphocytes

Highly purified preparations of Ca/Mg-dependent cell nuclear endonuclease have been obtained from human spleen lymphocytes. The purification was 2000-fold, and the enzyme was a polypeptide with a molecular weight of 57-54 kD. The study of its binding with monoclonal antibody enzyme, produced by various hybridoma strains obtained upon the immunization of mice with non-fractionated nuclear extract has shown that Ca/Mg-dependent endonuclease was the best expressed endonuclease of lymphocyte cellular nuclei. Possible production of some other cell nuclear endonucleases during processing of CH/Mg-dependent endonuclease is suggested.     

Study of heterogeneity of the chromatin endonuclease fragment by free flow electrophoresis]

The oligomer chromatin fragments relatively uniform in size (8--11 nucleosomes) were prepared by a short-term endonycleolysis. The heterogeneity of these fragments with respect to their electrophoretic mobility was revealed using free flow electrophoresis. The individual fragmentated chromatin subfractions were obtained. These subfractions differed in their protein and RNA content per DNA weight unit, in quantitative ratios of different zones of high molecular weight non-histone proteins and in thermal and alkaline denaturation kinetics. It was also found that the parameters investigated are correlated with electronegativeity of the fragmentated chromatin subfractions.     

Isolation of chromatin fragments rich in non-histone protein after endonuclease digestion of rat liver nuclei

Detergent-washed rat liver nuclei, prepared in the presence of a protease inhibitor, were incubated for up to 60 min at 37 °C. The action of endonucleases produced chromatin fragments which could be removed from the nuclei by extraction with 8 M urea 50 mM phosphate, pH 7.6, 15% of the total nuclear DNA being extracted. No DNA could be detected in this extract after incubation of nuclei at 4 °C. The chromatin fragments were sedimented by centrifugation at 90000 g or by chromatography on Sepharose 4B. The DNA fragment sizes were similar to those found in nucleosome particles but the protein/DNA ratio was approx. 5.3:1. The nuclei were prelabelled with [³H]tryptophan and 60% of the label present in the 8 M urea extract was found to sediment with the chromatin fraction. SDS polyacrylamide gel electrophoresis of the latter showed the presence, in addition to histones, of at least 25 polypeptide species of tightly bound non-histone proteins with molecular weights in excess of 30000.     

Changes in the compactness of nucleosome chromatin from the bovine liver during aging

Electrophoresis methods used to study the fragments of chromatin revealed under the effect of Ca,Mg-dependent endonuclease on it have permitted establishing that stability of chromatin to the nucleosome level increases with aging. Changes in the compactness of the chromatin structure with aging make the accessibility of the linker DNA to nuclease lower the size of DNA fragments corresponding to mononucleosomes increasing from 192 +/- 5 pairs of bases to 209 +/- 5 pairs. Stabilization of the chromatin structure begins from certain nucleosomes which become more compact with the age. When analyzing basic proteins of chromatin by electrophoresis in different systems no qualitative changes were found in the subfraction composition of histones with aging, that permits supposing nonhistone proteins of chromatin and histone H1 to participate in the change of the chromatin structure compactness with the age.     

Separation of satellite DNA chromatin and main band DNA chromatin from mouse brain.

Using restriction endonucleases which preferentially digest mouse main band DNA and leave satellite DNA intact, we have isolated highly purified chromatin fractions containing only mouse satellite or main band DNA. Following the digestion of mouse brain nuclei with EndoR Alu I, main band DNA chromatin is selectively extracted with 10mM Tris, 10mM EDTA. Satellite DNA chromatin is subsequently extracted from the nuclear pellet with Tris-3M urea and further purified on sucrose gradients. Chromatin extracted from digested nuclei with Tris-EDTA contains only main band DNA and has a molecular weight lower than 2 x 10(6). Chromatin fractions obtained from the lower regions of sucrose gradients of the Tris-Urea extracts contain 40--95% satellite DNA and have a molecular weight of 6 to 8 x 10(6). Both the satellite DNA and main band DNA chromatins contain all five histones and have a protein to DNA ratio of 1.3 to 1.     

Fractionation of chromosomal deoxyribonucleoproteins by partition in two-phase aqueous polymer systems

Deoxyribonucleoprotein (DNP)¹ prepared by shearing chromatin of mouse cells may be fractionated in 2-phase aqueous Dextran-polyethyleneglycol mixtures. A partial separation of DNPs with different non-histone protein/DNA ratios may be obtained in a single-step partition. Separation of a spectrum of fractions of DNP has been obtained by countercurrent distribution using the same 2-phase polymer system. DNP fractions which bear nascent RNA (representing approximately $\text{1}\text{3}$ of the total DNA) may be separated from the major fraction of DNP; they are found in the same region of the distribution pattern as DNP fractions with the highest non-histone protein/DNA ratio.     

On the role of Ca, Mg-dependent nuclease in the postirradiation degradation of chromatin in lymphoid tissues

The characteristics of the postirradiation degradation of chromatin in thymocytes in vivo were compared with the features of chromatin fragmentation in isolated thymocyte nuclei in vitro by endogenous chromatin-bound nucleases. Nuclease which degrades chromatin produces in vivo fragments of nucleosomal size; the double-strand breaks appear as the result of the accumulation of single-strand breaks with 3'-OH ends; the nuclease is inhibited by Zn2+ and DTNB and its activity is depressed by cycloheximide pretreatment. In experiments on in vitro degradation of chromatin in isolated thymocyte nuclei similar properties were observed for the Ca, Mg-dependent, but not for acid nuclease. The results bring further evidence of the involvement of an enzyme of the Ca, Mg-dependent nuclease-type in chromatin degradation in irradiated thymocytes.     

Interphase chromosomal deoxyribonucleoprotein isolated as a discrete structure from cultured cells

A new procedure is described for the preparation of interphase chromatin from cultured mouse cells (line P815). The primary objective of this procedure was to eliminate exchanges of histones between deoxynucleoprotein molecules; this objective is shown experimentally to have been attained. The chromatin is released from cells by the non-ionic detergent Nonidet P40 in medium of low ionic strength (0.1 mM-KNa₂PO₄), and may then be sedimented as a structure which conserves the general form and ultrastructural characteristics of chromatin within the cell. The nuclear envelope cannot be detected in these structures by electron microscopy, and their content of choline-containing phospholipids is less than 10% of that of nuclei. The maintenance of form in this structure must thus depend on properties of the chromatin itself, and possibly on the more compact peripheral chromatin.Soluble DNP² prepared by shearing these structures has the same relative contents of DNA, histones, non-histone proteins and RNA as DNP prepared by standard methods. Analyses by electrophoresis on polyacrylamide gels of the non-histone proteins reveals certain differences from the pattern of these proteins in DNP prepared by a salt precipitation method. The template activity for RNA synthesis, in the presence of Escherichia coli RNA polymerase of sheared, soluble DNP prepared by this procedure, is comparable to that of DNP prepared by other methods. However, in the absence of exogenous RNA polymerase the rate of RNA synthesis by structured (unsheared) chromatin is about ten times higher than the rate using sheared DNP.The rapid removal of the nuclear envelope in this lysis procedure allowed experimental examination of the origin of the histones and non-histone proteins of DNP. When DNP was prepared from a mixture of two populations of cells, one containing DNA distinguishable by a density label and the other containing radioactively labelled proteins, radioactive proteins were found exclusively in DNP of normal density, and not in dense DNP and vice versa. It is concluded that the proteins of DNP prepared in this way are not acquired during the preparation procedure but were already associated with DNA in vivo, and that other proteins are not bound non-specifically to DNA during the preparation of DNP. When a mixture of DNP molecules prepared, in this way is precipitated in 150 mm-NaCl and redissolved, some radioactively labelled histones migrate onto dense DNA molecules.This procedure is suitable for routine, quantitative isolation of chromosomal DNP from small numbers of cells; it is also applicable to cells of other cultured lines.     

Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease.

Treatment of either rat liver chromatin or intact nuclei with the enzyme staphylococcal nuclease results in the conversion of about half of the DNA to acid-soluble oligonucleotides. As previously described, mild digestion of nuclei results in the liberation of a series of nucleoprotein particles containing DNA fragments which are all integral multiples of a unit length DNA 185 base pairs in length. Analysis of the kinetics of appearance of these fragments suggests that at least 85% of the nuclear DNA is involved in the formation of the repeating subunit profile. More extensive digestion of nuclei however results in the generation of a series of eight unique DNA fragments containing 160 to 50 base pairs. The series of smaller molecular weight DNA is virtually identical with the profile obtained upon limit digestion of isolated chromatin. By velocity centrifugation we have obtained highly purified preparations of the monomeric nucleoprotein particle. Digestion of this monomeric subunit results in the solubilization of 46% of the DNA and analysis of the resistant DNA again reveals the set of eight lower molecular weight fragments. These data suggest that the initial site of nuclease cleavage in chromatin resides within the DNA bridging the repeating monomeric subunits. Further attack results in cleavage at a set of sites within the monomer liberating a pattern of smaller DNA fragments which probably represents the points of intimate contact between the histones and DNA.     

The effect of irradiation and alkylating agents on chromatin breakdown in normal and malignant lymphoid cells

Regularities of chromatin degradation in thymocytes and LS/BL tumor cells have been investigated. It has been shown that the rate of DNA degradation by Ca/Mg-dependent endonuclease in LS/BL tumor cells is 25 times lower than that in thymocytes, and radiation does not induce chromatin degradation. The alkylating agent TS 160 causes chromatin degradation in both LS/BL cells and thymocytes. In contrast to radiation TS 160 inhibits the endogenous chromatin degradation by Ca/Mg-dependent endonuclease in thymocytes.