Chromosome diminution in Ascaris suum. Two-fold increase of nucleosomal histone to DNA ratios during development

The swine intestinal nematode, Ascaris suum, eliminates chromatin material from its primordial somatic cells during early embryogenesis. A technique for isolation of nuclei from pre- and post-diminution stage embryos has been developed and these isolated nuclei were used in investigations of nuclear events during diminution. The amount of DNA per nucleus determined by diphenylamine assays and isotope dilutions was 0.66 pg and 0.29 pg in pre- and post-diminution nuclei, respectively. Thus, A. suum loses 56% of its nuclear DNA during diminution. The loss of nuclear DNA enabled in vivo examination of histone to DNA ratios as a function of changes in DNA quantities. Ascaris histones were identified by acid extractability and tryptic fingerprint comparison with rat liver histones. Measurement of histone quantities was accomplished using linearity of Coomassie blue binding to histones separated in dodecyl sulfate gels. Ascaris nucleosomal histones levels were relatively constant in pre- and post-diminution nuclei. However, nucleosomal histone to DNA ratios approximately doubled during diminution.     

Comparison of chromatin isolated from the Oo¨mycetesSaprolegnia andAchlya

The nucleosome DNA repeat length of chromatin fromSaprolegnia ferax was compared to that of the related Oo¨myceteAchlya ambisexualis and to DNA repeat lengths of chromatin from rat kidney and rat cerebral cortex neurons. The repeat length forSaprolegnia was 167 base pairs, consistent with reports of unusually short DNA repeat lengths in fungal chromatins. The acid-soluble nuclear proteins ofSaprolegnia were compared to those ofAchlya and to histones from the higher plant rye and from rabbit kidney. Both Oo¨mycetes contained acid-soluble nuclear proteins with electrophoretic mobilities identical to those of mammalian and higher plant histones H3 and H4. Also, both fungi contained a prominent unique band designated α, not seen in either the mammalian or plant preparations. Protein α has not been reported in other fungi; however, its presence in bothAchlya andSaprolegnia suggests it is an important nuclear protein in the Oo¨mycetes.     

Nuclear proteins and DNA in the myocardial myocytes in compensatory cardiac hypertrophy]

Using cytophotometry, the amount of DNA, total nuclear proteins and of histones were studied in the myocardial cells during days 21--36 of experimental compensatory hypertrophy of the heart (in rats). The enlargement of myocardial nuclei during the cardial hyperfunction was accompanied by accumulation of total nuclear protein, in particular, the histone fraction, without distinct changes in DNA. Analysis of correlations between nuclear proteins and DNA in the myocardial cells allows to reveal a delayed accumulation of histones in the big and gigantic nuclei, with a superfluous increase in non-histone nuclear proteins. In middle-sized nuclei, non-histone proteins have little changes against intensive accumulation of histones.     

Cytochemical studies on the protamine-type protein transition in sperm nuclei after fertilization and the early embryonic histones of Urechis caupo.

Cytochemical staining characteristics of nuclear histones during postfertilization maturation division and various early embryonic stages in Urechis have been studied. The transition of protamine-type protein to adult histones in the sperm nucleus is accomplished by 15 min after entrance into the egg cytoplasm. Newly synthesized egg proteins migrate into enlarging male and female pronuclei after this transition, followed by pronuclear DNA synthesis and fusion. The shift from protamine-type protein to adult histones, which occurs in the absence of RNA synthesis during the postfertilization maturation division of the egg, may be one of the processes involved in the normal structural reorganization of chromosomes. Such a reorganization is likely to be a prerequisite for chromosome replication and mitosis. No qualitative differences are detected in the stainability of histones of unfertilized eggs and embryos at the cleavage and later stages of development.     

Nepsilon-formylation of lysine is a widespread post-translational modification of nuclear proteins occurring at residues involved in regulation of chromatin function

Post-translational modification of histones and other chromosomal proteins regulates chromatin conformation and gene activity. Methylation and acetylation of lysyl residues are among the most frequently described modifications in these proteins. Whereas these modifications have been studied in detail, very little is known about a recently discovered chemical modification, the N^ε-lysine formylation, in histones and other nuclear proteins. Here we mapped, for the first time, the sites of lysine formylation in histones and several other nuclear proteins. We found that core and linker histones are formylated at multiple lysyl residues located both in the tails and globular domains of histones. In core histones, formylation was found at lysyl residues known to be involved in organization of nucleosomal particles that are frequently acetylated and methylated. In linker histones and high mobility group proteins, multiple formylation sites were mapped to residues with important role in DNA binding. N^ε-lysine formylation in chromosomal proteins is relatively abundant, suggesting that it may interfere with epigenetic mechanisms governing chromatin function, which could lead to deregulation of the cell and disease.     

Cloning and Functional Analysis of Histones H3 and H4 in Nuclear Shaping during Spermatogenesis of the Chinese Mitten Crab,Eriocheir sinensis

During spermatogenesis in most animals, the basic proteins associated with DNA are continuously changing and somatic-typed histones are partly replaced by sperm-specific histones, which are then successively replaced by transition proteins and protamines. With the replacement of sperm nuclear basic proteins, nuclei progressively undergo chromatin condensation. The Chinese Mitten Crab (Eriocheir sinensis) is also known as the hairy crab or river crab (phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Grapsidae). The spermatozoa of this species are aflagellate, and each has a spherical acrosome surrounded by a cup-shaped nucleus, peculiar to brachyurans. An interesting characteristic of the E. sinensis sperm nucleus is its lack of electron-dense chromatin. However, its formation is not clear. In this study, sequences encoding histones H3 and H4 were cloned by polymerase chain reaction amplification. Western blotting indicated that H3 and H4 existed in the sperm nuclei. Immunofluorescence and ultrastructural immunocytochemistry demonstrated that histones H3 and H4 were both present in the nuclei of spermatogonia, spermatocytes, spermatids and mature spermatozoa. The nuclear labeling density of histone H4 decreased in sperm nuclei, while histone H3 labeling was not changed significantly. Quantitative real-time PCR showed that the mRNA expression levels of histones H3 and H4 were higher at mitotic and meiotic stages than in later spermiogenesis. Our study demonstrates that the mature sperm nuclei of E. sinensis contain histones H3 and H4. This is the first report that the mature sperm nucleus of E. sinensis contains histones H3 and H4. This finding extends the study of sperm histones of E. sinensis and provides some basic data for exploring how decapod crustaceans form uncondensed sperm chromatin.     

THE SYNTHESIS OF ACIDIC CHROMOSOMAL PROTEINS DURING THE CELL CYCLE OF HELA S-3 CELLS : I. The Accelerated Accumulation of Acidic Residual Nuclear Protein before the Initiation of DNA Replication

The synthesis and accumulation of acidic proteins in the tightly bound residual nuclear fraction goes on throughout the cell cycle of continuously dividing populations of HeLa S-3 cells; however, during late G₁ there is an increased rate of synthesis and accumulation of these proteins which precedes the onset of DNA synthesis. Unlike that of the histones, whose synthesis is tightly coupled to DNA replication, the synthesis of acidic residual nuclear proteins is insensitive to inhibitors of DNA synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acidic residual nuclear proteins shows different profiles during the G₁, S, and G₂ phases of the cell cycle. These results suggest that, in contrast to histones whose synthesis appears to be highly regulated, the acidic residual proteins may have a regulatory function in the control of cell proliferation in continuously dividing mammalian cells.     

Changing patterns of nucleic acids, basic and acidic proteins in generative nuclei during pollen germination and pollen tube growth in Hippeastrum belladona

Generative and vegetative nuclei of mature and germinated pollen grains from Hippeastrum belladonna were separated in a continuous Ficoll gradient. Less than 3% contamination was observed between the generative and vegetative nuclear fractions. The vegetative nuclei were composed of two populations; the larger population consisted of nuclei with 1C levels of DNA and the smaller with 2C levels. The generative nuclei consisted of a homogeneous population composed of nuclei possessing 2C levels of DNA. Histone synthesis did not occur in vegetative nuclei. Changes appeared in the gel-electrophoretic banding patterns of the F1 histones of vegetative nuclei during germination. Changes were not observed in the generative nuclei. A reduction of general proteins and RNA was observed in vegetative nuclei by 20 h of germination. The phenol-soluble nuclear proteins of vegetative nuclei revealed transitions in electrophoretic banding patterns during pollen germination that were greater than those shown by the histones. These changes in the PSNP primarily involved reduced concentrations of certain proteins rather than synthesis of new ones. However, a new band was observed in the electrophoretic pattern of the PSNP of vegetative nuclei after 12 h of pollen tube growth. No transition was seen in the PSNP of generative nuclei during pollen germination and tube growth. The regulatory role of the PSNP in cell differentiation is discussed in the light of these findings.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Evidence for attachment of interphase chromatin to the nuclear matrix via matrix-bound nucleosomes

Chromatin structure has been studied in the sites of attachment to the nuclear matrix in interphase mouse liver and spleen nuclei. The patterns of fragmentation of the DNA belonging to these sites (0.3-2% of total DNA in spleen and liver, respectively) with staphylococcal nuclease and DNAase I were very close to those of usual nucleosomal chains. Moreover, the nuclear matrix preparations contained all five major histones, including H1, in almost stoichiometric amounts. The histone/DNA ratios for the matrix were also similar to those found in nuclei. These findings and the size of the matrix-protected DNA indicated that interphase chromatin was attached to the nuclear matrix via matrix-bound nucleosomes and, to a much lesser extent, oligonucleosomes up to 5-6 units long. Two-dimensional electrophoretic separation of the matrix-bound histones revealed that modifications of histone H1 and, probably, of other histones were distinguished from those in bulk chromatin. Study of binding of exogenously added labeled histone octamers or mononucleosomal size DNA to nuclear matrix excluded the possibility of their artifactual trapping during the isolation procedure.     

Molecular characterization of Ascaris suum DNA and of chromatin diminution

A technique for the extraction of pure somatic (post-diminution) and germ line (pre-diminution) DNA from the parasitic nematode Ascaris is described. Uncontaminated post- and pre-diminution DNAs were sheared and reassociated to different C₀t values. Computer analysis of the complete reassociation kinetics determined that 33% of the germ line genome is eliminated during the process of chromatin diminution. The eliminated DNA is comprised of repetitive and unique sequences in an approx. 1:1 ratio.     

Triiodothyronine nuclear receptor and the role of non-histone protein factors in in vitro triiodothyronine binding

The rat liver triiodothyronine (T3) nuclear receptor rapidly looses, after a partial purification from the nuclear extract, its ability to bind T3. We previously reported that histones, in the presence of DNA, could protect against inactivation enhancing the T3 binding site concentration and maintaining the high affinity for T3. A nuclear fraction discarded during the receptor purification (fraction A) was also found able to restore T3 binding and was analyzed. As histones + DNA, fraction A stabilized the T3 binding site from irreversible inactivation during incubation with T3, increasing its concentration while keeping the same high affinity for T3. It was active even at relatively high receptor concentration, appeared slightly more active than histones (+ DNA) in the same protein concentration range (up to 50-fold increment of T3 binding at the optimal concentration of 25 micrograms/ml) and was unaffected or slightly inhibited by DNA. Other proteins (ovalbumin, soybean trypsin inhibitor, RNAase) and rat liver cytosol were several times less effective, suggesting a major role of some nuclear constituents. The active factors in fraction A essentially belong to non-histone nuclear proteins. Fraction A was found heterogeneous regarding the molecular size and pHi of the active factors, the existence of subfractions more active on a protein concentration basis being suggested but not yet clearly evidenced. Efficient in vitro T3 binding to the isolated T3 nuclear receptor thus depends on the presence of several different nuclear constituents, histones + DNA or some non-histone proteins. Whether interactions with these constituents could modulate T3 binding within the nucleus remains to be elucidated.     

ANTIGEN-INDUCED CHANGES IN LYMPHOID CELL HISTONES : II. Regional Lymph Nodes

The nuclear histones of control and antigen-affected lymphocytes and thymocytes were measured in terms of their intensity of staining with ammoniacal-silver (A-S) and alkaline fast green (FG). Subcutaneous injection of full strength and doubly concentrated tetanus toxoid into the upper forelimb was followed by an acute decrease in the intensity of the FG and A-S staining of the thymocyte nuclear histones. Such changes in staining were delayed in the nuclear histones of the regional axillary lymph nodes. In contrast, the injection of a 1:100 dilution of tetanus toxoid caused an acute decrease in staining of the nuclei of the regional lymph node lymphocytes but only minimal changes in the thymocytes. An acute decrease in staining of the lymph node lymphocytes also occurred when heterologous serum proteins were used as the antigen. Prior administration of cortisone was found to inhibit the antigen-induced changes in the staining of lymphocyte nuclear histones. No such inhibitions occurred after the administration of saline solution. The findings suggest that the nuclear histones play a significant role in the immunological competence of lymphoid cells. However, additional studies are needed to define the biochemical basis of the observed changes as well as the mechanisms whereby antigen-induced changes in nuclear histones participate in the complex cellular and serological alterations which are evoked by antigens, viz. plasma cell formation and antibody synthesis.     

Nuclear reassemblyin vitro is independent of nucleosome/chromatin assembly

It was show11 that nuclear reassembly was induced by small pieces of DNA fragments in cell-free extracts ofXenopus. In an attempt to learn the relationship between the nuclear reassembly and nucleosome/chromatin assembly, limited amounts of CM-Cellulose are used to eliminate the capacity of the egg extract S-150 to assemble chromatin. while the forming of nucleosomes is checked with DNA supercoiling by plasmid DNA pBR322 incubated in the extract, and further analysed by micrococcal nuclease digestion. This depleted extract is then used to induce nuclear reassembly around demembraned sperms with membrane vesicles. It is found that CM-Cellulose depletes histones H2A and H2B efficiently and blocks the assembly of nucleosomes, the demembraned sperms are yet reconstituted into nuclei in the treated S-150, although the chromatin in reassembled nuclei does not produce protected DNA fragments when digested with micrococcal nuclease. It suggests that in the cell-free system ofXenopus, DNA can be formed into nuclei without assembly of nucleosomes or chromatin.     

Proteins associated with mitochondrial DNA protect it against the action of X-rays and hydrogen peroxide

It has been suggested in a number of investigations that the high vulnerability of mitochondrial DNA to reactive oxygen species and other damaging agents is due to the absence in mitochondria of histones complexed with DNA. In the present study it was shown that DNA-binding proteins of mitochondrial nucleoids were able to shield mitochondrial DNA from X-ray radiation and hydrogen peroxide, as nuclear histones did. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver cells. The degree of damage to or protection of mitochondrial DNA was assessed from the yield of its PCR amplification product. The in vitro experiments demonstrated that mouse mitochondrial DNA, when in complex with mitochondrial nucleoids or nuclear histones, was damaged much less by radiation and/or hydrogen peroxide than in the absence of these proteins and histones. No significant difference between mitochondrial nucleoid proteins and nuclear histones was revealed in their efficiency to protect mitochondrial DNA from the damaging effect of radiation and hydrogen peroxide. It is likely that the nucleoid proteins in the mitochondria shield mitochondrial DNA against the attack of reactive oxygen species, thus significantly decreasing the level of the oxidative damage to mitochondrial DNA.     

Effects of thyrotropin on thyroid chromatin: Enhanced sensitivity to micrococcal nuclease and increased nuclear protein phosphorylation

Thyroid slices were incubated with or without TSH for 2 or 5 h. Nuclei were then prepared, subjected to mild digestion with micrococcal nuclease, and centrifuged at 1200 × g. The amount of DNA in 1200 × g supernatants was increased by TSH at 5 h, but not at 2 h. In parallel studies, thyroid slices were incubated with ³²P_i and labeling of acid-soluble nuclear proteins was examined. TSH-dependent increases in labeling of histones H1 and H3, and of the high mobility group protein HMG 14, were observed at 2 h; however, there were no apparent changes in TSH-dependent labeling between 2 and 5 h, in nuclease-sensitive or in bulk chromatin. These results suggest that the observed TSH-dependent changes in the micrococcal nuclease-sensitivity of thyroid nuclear chromatin were not induced directly by changes in the phosphorylation of the histones or HMG 14.     

Cytochemistry of histones throughout the division cycle in meristematic cells

Summary We have been able to follow the increase in nuclear histones along the division cycle, using a natural synchronous meristematic cell population labelled by caffeine as binucleate. We have thus found a parallel increase in DNA and histone along the S period. Some increase in nuclear histones during the G₁ and G₂ has also been detected. At the same time a high rise of stainable histone in the nuclei in prophase has been found.Changes in the composition of nuclear histones have been followed counting the percentage of arginine-rich histones in the total histone content. Two peaks located at the beginning and at the end of the S period as well as a sharp descent of the percentage of arginine-rich histone in the prophasic nucleus have been found.     

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.     

Flow cytometric and biochemical analysis of dose-dependent effects of sodium butyrate on human endometrial adenocarcinoma cells.

Sodium butyrate (SB) treatment was previously shown to produce seven-fold increases in estrogen hormone receptor binding sites of human endometrial adenocarcinoma (IK) cells. Flow cytometric analysis and histone gel electrophoresis were used to examine cell cycle, cell metabolism, and nuclear histone fractions in IK cells treated with different concentrations of SB. SB-treated cells stained with fluorochromes specific for DNA, RNA, or general protein were analyzed by flow cytometry (FCM). Changes in accessibility to three DNA stains and gel electrophoresis were used to analyze rearrangements in chromatin structure. SB caused an accumulation of cells in the G1 phase and inhibited DNA synthesis, but not cellular levels of RNA and protein. Hoechst accessibility to A-T rich regions on DNA was dramatically increased after removal of SB. H1 histones were dephosphorylated and core histones were acetylated during SB-treatment. Information obtained in these studies may be useful for correlating cellular and biochemical events with SB-induced increases in nuclear steroid hormone binding sites.