Chromatin nu bodies: isolation, subfractionation and physical characterization.

Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study.     

Ultrastructural features of chromatin nu bodies

Spread chromatin fibers and isolated chromatin fragments prepared from chicken erythrocyte nuclei were stained with dilute aqueous uranyl acetate. High-resolution electron micrographs reveal two new morphological features exhibited by many of the chromatin nu bodies: (a) lateral association of the nu body with the connecting strand, and (b) a centrally stained spot approximately 15 A wide, possibly corresponding to a hole or crevice within the nu body.     

Conformational states of chromatin nu bodies induced by urea.

Monomer chromatin nu bodies (nu1) from chicken erythrocyte nuclei were exposed to 0-10 M urea plus 0.2 mM EDTA (PH 7). Alterations in nu1 conformation were examined using hydrodynamic methods (i.e., S, eta, and (formula: see text)), thermal denaturation, circular dichroism, reactivity of histone thiol groups to N-ethyl maleimide, and electron microscopy. The two domains of a nu body (i.e., the DNA-rich shell and the protein-rich core) aeared to respond differently to the destabilizing effects of increasing urea; DNA conformation and stability exhibited noncooperative changes; the core protein structure revealed cooperative destabilization between 4 and 7 M urea. Companion studies on the conformation of the inner histone "heterotypic tetramer" also revealed cooperative destabilization with increasing urea concentration.     

Spatial resolution of neuromagnetic records: theoretical calculations in a spherical model

Spatial resolution of magnetoencephalography (MEG) was studied by computer simulations using a spherical conductor model for the head. The accuracy obtainable in the absolute location of a dipole was found by calculating the confidence limits for source location in 3 dimensions. The accuracy in determining the relative locations of two sources was estimated by calculating the smallest shift in source location that could be detected with statistical significance. The results were used to illustrate the dependence of spatial resolution on several factors including noise, source depth, source strength, flux transformer configuration and the choice of the measurement locations. Under optimal conditions, separations of a couple of millimeters in superficial non-simultaneous sources can be detected, whereas for deeper sources the resolution is worse.     

Bacteriophage-associated spherical bodies in Bacteroides fragilis.

Unique spherical bodies with multilayered walls were observed by electron microscopy in cells of a single strain of Bacteroides fragilis subsp. fragilis. Phage-like particles were present in the same cells, both free in the cytoplasm and within the spheres. The proportion of cells containing the phage-associated spherical structures ranged from less than 0.01% to about 7% depending on the culture conditions. Phage particles of morphological type B and spherical bodies were also found free in the medium surrounding the cells. Spherical bodies with discontinuities in their walls, through which phage-like particles sometimes appeared to be escaping, were also found both intra- and extracellularly. The biological significance of these distinctive spherical structures is a matter of conjecture.     

Off-center spherical model for dosimetry calculations in chick brain tissue

This paper presents calculations for the electric field and absorbed power density distribution in chick brain tissue inside a test tube, using an off-center spherical model. It is shown that the off-center spherical model overcomes many of the limitations of the concentric spherical model, and permits a more realistic modeling of the brain tissue as it sits in the bottom of the test tube surrounded by buffer solution. The effect of the unequal amount of buffer solution above the upper and below the lower surfaces of the brain is analyzed. The field distribution is obtained in terms of a rapidly converging series of zonal harmonics. A method that permits the expansion of spherical harmonics about an off-center origin in terms of spherical harmonics at the origin is developed to calculate in closed form the electric field distribution. Numerical results are presented for the absorbed power density distribution at a carrier frequency of 147 MHz. It is shown that the absorbed power density increases toward the bottom of the brain surface. Scaling relations are developed by keeping the electric field intensity in the brain tissue the same at two different frequencies. Scaling relations inside, as well as outside, the brain surface are given. The scaling relation distribution is calculated as a function of position, and compared to the scaling relations obtained in the concentric spherical model. It is shown that the off-center spherical model yields scaling ratios in the brain tissue that lie between the extreme values predicted by the concentric and isolated spherical models.     

Cooperative alignment of nu bodies during chromosome replication in the presence of cycloheximide

50% of control DNA is resistant to staphylococcal nuclease after digestion in isolated nuclei, while only 25% of the labeled DNA made in the presence of cycloheximide is resistant to nuclease. Nevertheless, cycloheximide DNA is folded into normal chromosomal subunits as evidenced by the observation that it generates nuclese limit-digest DNA fragments that are indistinguishable from controls. These results indicate that cycloheximide chromatin is associated with half the number of normal nu bodies. These nu bodies are probably recycled from the parental chromosome. Partial nuclease digestion of cycloheximide chromatin reveals that a normal pattern of monomer and multimer DNA fragments is generated up to octamers. The data are consistent with the idea that in the presence of cycloheximide, recycled parental histones become cooperatively aligned along the daughter double helices.     

Crowding effects on the formation and maintenance of nuclear bodies: insights from molecular-dynamics simulations of simple spherical model particles

The physics of structure formation and maintenance of nuclear bodies (NBs), such as nucleoli, Cajal bodies, promyelocytic leukemia bodies, and speckles, in a crowded nuclear environment remains largely unknown. We investigate the role of macromolecular crowding in the formation and maintenance of NBs using computer simulations of a simple spherical model, called Lennard-Jones (LJ) particles. LJ particles form a one-phase, dilute fluid when the intermolecular interaction is weaker than a critical value, above which they phase separate and form a condensed domain. We find that when volume-exclusive crowders exist in significant concentrations, domain formation is induced even for weaker intermolecular interactions, and the effect is more pronounced with increasing crowder concentration. Simulation results show that a previous experimental finding that promyelocytic leukemia bodies disappear in the less-crowded condition and reassemble in the normal crowded condition can be interpreted as a consequence of the increased intermolecular interactions between NB proteins due to crowding. Based on further analysis of the simulation results, we discuss the acceleration of macromolecular associations that occur within NBs, and the delay of diffusive transport of macromolecules within and out of NBs when the crowder concentration increases. This study suggests that in a polydisperse nuclear environment that is enriched with a variety of macromolecules, macromolecular crowding not only plays an important role in the formation and maintenance of NBs, but also may perform some regulatory functions in response to alterations in the crowding conditions.     

染色质结构之美——染色质可及性

染色质可及性(chromatin accessibility)作为一种衡量染色质结合因子与染色质DNA结合能力高低的染色质属性,是评价染色质结构稳态的重要指标之一,在多种细胞核进程中扮演重要角色,包括基因转录调控以及DNA损伤修复等。该属性的异常调控与多种疾病的发生发展密切相关,包括肿瘤以及神经退行性疾病等。对于该属性探究已经成为生命科学与疾病领域的热点。伴随越来越多的新技术应运而生,例如染色质构象捕获技术、高通量测序技术以及两种技术的结合等。随着技术的进步,多种参与调控染色质可及性的因素被发现和总结,包括核小体占位、组蛋白修饰以及非编码RNA等。多项大规模的染色质组学数据绘制了多种疾病的染色质可及性图谱,为揭示疾病的发生发展与染色质可及性之间的关系提供了数据支持。同时,随着单细胞染色质可及性测序技术的发展,实现了对细胞类型染色质层面的划分,弥补了单纯依赖基因表达划分细胞类型的不足。本文将从染色质的组成与可及性、影响染色质可及性的因素、染色质可及性的检测方法,以及染色质可及性与癌症的关系等方面简要阐述染色质可及性的研究进展。     

Density functional calculations on model tyrosyl radicals.

A gradient-corrected density functional theory approach (PWP86) has been applied, together with large basis sets (IGLO-III), to investigate the structure and hyperfine properties of model tyrosyl free radicals. In nature, these radicals are observed in, e.g., the charge transfer pathways in photosystem II (PSII) and in ribonucleotide reductases (RNRs). By comparing spin density distributions and proton hyperfine couplings with experimental data, it is confirmed that the tyrosyl radicals present in the proteins are neutral. It is shown that hydrogen bonding to the phenoxyl oxygen atom, when present, causes a reduction in spin density on O and a corresponding increase on C4. Calculated proton hyperfine coupling constants for the beta-protons show that the alpha-carbon is rotated 75-80 degrees out of the plane of the ring in PSII and Salmonella typhimurium RNR, but only 20-30 degrees in, e.g., Escherichia coli, mouse, herpes simplex, and bacteriophage T4-induced RNRs. Furthermore, based on the present calculations, we have revised the empirical parameters used in the experimental determination of the oxygen spin density in the tyrosyl radical in E. coli RNR and of the ring carbon spin densities, from measured hyperfine coupling constants.     

Studies on the mode of segregation of histone nu bodies during replication in HeLa cells

Two models were tested for the mode of distribution of histone nu bodies at the replication fork. The replication fork was labeled by brief incubation of cells with ³H-thymidine. Nuclei were isolated and digested with low levels of micrococcal nuclease, and the kinetics of cleavage of the pulse-labeled chromatin DNA were compared to the kinetics of cleavage of parental chromatin DNA. In chromatin labeled for 30 sec to 10 min, the rate of cleavage of the pulse-labeled region into monomeric nu body-sized units exceeded the rate of cleavage of parental chromatin by a factor of 2, but did not approach the predicted value of 5–6 for random segregation. This value dropped to 1.6 in 15 min and was euivalent to parental chromatin in 20 min labeling experiments. DNA synthesized in the presence of cycloheximide was also digested at twice the rate of parental chromatin DNA.A Poisson analysis of the kinetics of cleavage by micrococcal nuclease further confirmed these observations. The predicted difference in the rate of production of monomeric, dimeric, and trimeric deoxyribonucleoprotein units was very similar to the experimental values of both total chromatin and nascent chromatin. Thus the nu body spacings in newly replicated chromatin closely approximate those in parental chromatin.These results agree well with a conservative or nondispersive model of nucleosome distribution in which the proteins are associated with one of the two daughter chromosomes during replication.     

A cell population in nu/nu spleen can prevent generation of cytotoxic lymphocytes by normal spleen cells against self antigens of the nu/nu spleen.

Spleen cells from athymic nu/nu mice contain two kinds of physically separable active cells that can have very different effects on the generation of CL (cytotoxic lymphocytes) by normal LN cells in an in vitro response against allogeneic stimulator cells. They can provide an accessory cell required for the activation of CLP (cytotoxic lymphocyte precursor cells) which need not be H-2 identical to the CLP and will function normally even when H-2 identical to the stimulator cells. They can also provide a suppressor cell that prevents the activation of CLP that can recognize the H-2 of the nu/nu mouse. Thus, with A, B, and C to represent three H-2 differnt mouse strains, a culture containing CLP from strain A and nu/nu spleen cells from strain B or strain (A x B)F1 will produce CL against strain C or (A x C)F1 stimulator cells but not against strain B or strain (A x B)F1 stimulator cells unless the suppressor cell is first removed. It is proposed that the in vivo role of the suppressor cell in a normal mouse is to prevent the activation of CLP reactive against self.     

Magnetic resonance behavior of normal and diseased lungs: spherical shell model simulations.

The alveolar air-tissue interface affects the lung NMR signal, because it results in a susceptibility-induced magnetic field inhomogeneity. The air-tissue interface effect can be detected and quantified by measuring the difference signal (Delta) from a pair of NMR images obtained using temporally symmetric and asymmetric spin-echo sequences. The present study describes a multicompartment alveolar model (consisting of a collection of noninteracting spherical water shells) that simulates the behavior of Delta as a function of the level of lung inflation and can be used to predict the NMR response to various types of lung injury. The model was used to predict Delta as a function of the inflation level (with the assumption of sequential alveolar recruitment, partly parallel to distension) and to simulate pulmonary edema by deriving equations that describe Delta for a collection of spherical shells representing combinations of collapsed, flooded, and inflated alveoli. Our theoretical data were compared with those provided by other models and with experimental data obtained from the literature. Our results suggest that NMR Delta measurements can be used to study the mechanisms underlying the lung pressure-volume behavior, to characterize lung injury, and to assess the contributions of alveolar recruitment and distension to the lung volume changes in response to the application of positive airway pressure (e.g., positive end-expiratory pressure).     

Chromatin replication: Finding the right connection.

Nucleosomes are preferentially assembled on replicating DNA by chromatin assembly factor 1; recent studies have shown that replicated DNA is marked for assembly into chromatin by the replication-fork-associated protein PCNA.     

Quantum-chemical and empirical calculations on phospholipids: V. Conformational calculations on model headgroups with varying ammonium group methylation

Using empirical 0-1-6-12 atom-atom potential functions and the PCILO method the conformational properties of anhydrous and hydrated model headgroups with varying ammonium group methylation were investigated. With a phosphoryl gauche^(?)-gauche^(?) conformation the torsion angle α₄ lies in the region of 180°–300° for all compounds. Torsion angles α₄ = 300° ? 100° are forbidden due to intramolecular sterical hindrance. The torsion angles α₅ and α₆ are influenced by the stage of ammonium group methylation and bound water molecules. The minima of energy with respect to α₅ were found at an ± syn-clinal (and for PC and DMPE + H₂O at an anti-periplanar) conformation.     

Chromatin models. The ionic strength dependence of model histone-DNA interactions: circular dichroism studies of lysine-leucine polypeptide-DNA complexes.

The ionic strength dependence of the complexes between DNA and both random, (Lysx, Leuy)n, and block copolymers, (Lysx)n(Leuy)m, of lysine and leucine, with different amino acid compositions, was studied using circular dichroism (CD) as the probe to detect conformational differences in these complexes relative to native DNA. It was found that the CD spectra of complexes of both the random (Lys84, Leu16)n and block (Lys85)n(Leu15)m copolymers with DNA show a very sharp ionic strength dependence. The maximum altered CD spectrum for the complexes with the block copolymer was found to occur at the same ionic strength as that for poly(L-lysine)-DNA complexes, while the maximum CD change for the random copolymer complex occurred at a slightly lower ionic strength. This sharp dependence of the CD change on the ionic strength was found to be independent of the polymer/DNA ratio, r, for each individual copolymer. The CD spectra for these complexes at optimum NaCl concentration resemble those of the psi spectra of DNA [Jordan, C. F., Lerman, L.S., and Venable, J.H. (1972), Nature (London), New Biol. 236, 67]. The complexes of the random copolymer, (Lys68, Leu32)n, with DNA (r=0.25) at 0.15 M NaCl and below have CD spectra that resemble the A-form DNA spectra. The ionic strength dependence of the CD spectra of this complex is not as sharp as observed with the above polymers and has a broad positive plateau. It is suggested that both the CD spectra of these complexes reflect the phenomena of DNA condensation into a higher order asymmetric structure (folded and compact). The block copolymer, (Lys77)n(Leu23)m, complexes with DNA show very slight alterations in the CD spectra, with respect to native DNA. It appears that the long Leu sequence at one end of such copolymers may be unpropitious for causing the polypeptide-DNA complex to condense into a higher order asymmetric structure. Thus the importance of the distribution of hydrophobic residues, in the copolypeptides of Lys, is shown for causing condensation of complexes with DNA. The relevance of these findings to histone-DNA complexes in chromatin is discussed.     

Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells.

Spleens and lymph nodes of nu/nu, congenitally athymic, mice have 16–32% and 72–76%, respectively, of large, brain-associated-T (BAT) antigen positive cells. These BAT positive large cells were isolated from nu/nu spleens with a fluorescence-activated cell sorter (FACS-II) and then cultured on thymic reticuloepithelial cell (TRC) monolayers. Both concanavalin A and mixed lymphocyte reactivity was induced during this culture period. We concluded that these BAT positive cells are pre-T cells which can he induced to at least some T cell functions by an inductive factor produced by TRC.