DNA folding by histones: the kinetics of chromatin core particle reassembly and the interaction of nucleosomes with histones.

The kinetics of the chromatin core particle reassembly reaction in solution were quantitatively studied under conditions such that nucleohistone aggregation did not occur. Core particles, salt-jumped rapidly by dilution from 2.5 m-NaCl (in which DNA and histones do not interact) to 0.6 m-NaCl (in which core particles are nearly intact), reassemble in two distinct time ranges. Approximately 75% of the DNA refolds into core particle-like structures “instantaneously” as measured by several physical and chemical techniques with dead times in the seconds to minutes time range. The remaining DNA refolds with relaxation times ranging from 250 minutes at 0 °C to 80 minutes at 37 °C; this slow effect cannot be attributed to sample heterogeneity. The fraction of slowly refolding DNA and the slow relaxation time are independent of the core particle concentration. Transient intermediates present during the slow phase of refolding were identified as free DNA and core particle-like structures containing excess histone. Mixing experiments with DNA, histones, and core particles showed that core particle-histone interactions are responsible for the slow kinetics of DNA refolding. Upon treatment of reassembling core particles with the protein crosslinking reagent, dimethylsuberimidate, the slow phase of the reassembly reaction was arrested and a 13 S particle containing DNA and two octamers of histone was isolated. Consistent with the nature of this kinetic intermediate, it is shown that in 0.6 m-NaCl, core particles co-operatively bind at least one additional equivalent of histones with high affinity in the form of excess octamers. Also, core particles continue to adsorb considerably more histones with a weaker association constant of the order 10⁵m^?1 (in units of octamers) to a maximum value of 12 ± 2 equivalents (octamers) per core particle. The sedimentation coefficient increases with the two-thirds power of the molecular weight of the complex, as it would in the case of clustered spheres.A reassembly mechanism consistent with the data is presented, and other simple mechanisms are excluded. In the proposed mechanism, core particles reassemble very rapidly and compete effectively with DNA for histones such that approximately one-third of the particles initially formed are complexed with an excess octamer of histones, and 25% of the total DNA remains uncomplexed. The amount of this unusual reaction intermediate decays slowly to an equilibrium value of about 10%, thereby leaving 9% of the total DNA uncomplexed. Approximate values are calculated for the free energies, rate constants, and two of the activation energies which characterize this migrating octamer mechanism. This mechanism provides a means whereby histone octamers can be temporarily stripped off DNA at a modest free energy cost, approximately 2.6 kcal per nucleosome. Also, the properties of excess histone adsorption by chromatin and octamer migration suggest an efficient mechanism, consistent with observations by others, for nucleosome assembly in vivo during replication.     

The sea urchin (Sphaerechinus granularis) codes different H2B histones to assemble sperm and embryo chromatin.

Histones from sperm and gastrula nuclei of the sea urchin Sphaerechinus granularis were analyzed by three different electrophoretic methods, stained with specific color reactions and compared with one another. The histones of the two cell types showed identical properties in none of the three analytical methods employed but the greatest differences involved the H2B type of histones. H2B from embryo chromatin had properties similar to those of the corresponding molecule from calf thymus. In the sperm chromatin no such molecule was observed but two additional ones were found that differed from embryo H2B in size, charge and specific colour reactions. The differences were such that different genes may be postulated for the synthesis of the H2B histones found in sperm and in embryo.     

Laser Raman identification of an interaction site on DNA for arginine containing histones in chromatin.

Comparisons of the Raman spectra of DNA, chromatin, and complexes of DNA with poly-L-arginine and N-α-acetylarginine have been made. Both in native chromatin and in complexes of DNA with the arginine derivatives there is a marked decreased in the Raman intensity of the 1490±2 cm^?1 band due to guanine. Considerable evidence is presented to show that a decrease in the intensity of the 1490 cm^?1 Raman band of quanine in DNA is strong indication of a hydrogen bond being attached to the N-7 position of quanine. A specific model is presented for the interaction of the arginine residues with the guanine residues in the major groove of DNA. The Raman frequency of the histone Amide 1 band indicates that these protein molecules have a high α-helical content while the phosphate diester stretch frequency of the DNA shows the DNA to be in the B-family.     

Sperm histones and chromatin structure of the "primitive" sea urchin Eucidaris tribuloides

The "primitive" sea urchin Eucidaris tribuloides resembles the advanced sea urchins (euechinoids) in many respects, yet some features of its biochemistry and morphogenesis are more similar to other echinoderms such as starfish or sea cucumbers. Two unique characteristics of the sperm chromatin of all known euechinoids are an extremely long average nucleosomal repeat length and the presence of two male germ-line-specific histone variants, Sp H1 and Sp H2B. Histone composition and nucleosomal repeat length of the sperm chromatin of Eucidaris were compared to those of several euechinoids and a starfish. Eucidaris sperm chromatin contained large H1 and H2B histone variants typical of euechinoids. The H1 was about nine amino acids smaller than Sp H1 of the advanced urchin Strongylocentrotus purpuratus. Its Sp H2B molecules were the same size as in the euechinoids. Peptide maps showed that N-terminal regions of Sp H1 and Sp H2B contained repeating basic amino acid motifs characteristic of euechinoids. The smaller size of Eucidaris H1 is accounted for by a smaller C-terminal region. The repeat length of Eucidaris sperm chromatin was slightly shorter than that of two euechinoids, but significantly larger than starfish, which lacks a large H2B. The Sp H2B gene of Eucidaris was expressed during spermatogenesis in the same cell types as for S. purpuratus. Thus Sp histone subtype expression and chromatin structure in this distantly related echinoid closely resemble the euechinoids. The presence of an Sp H2B and a very long repeat length appear to be characteristic of the echinoids only.     

Photochemical cross-linking of histones to DNA nucleosomes.

Ultraviolet (UV)-induced cross-linking was utilized in order to identify histone-DNA interacting regions in the chromatin repeating unit. Fractionated mononucleosomes which contained 185 base pairs of DNA and a full complement of the histones, including histone H1, were irradiated with light of lambda greater than 290nm in the presence of a photosensitizer. Equimolar amounts of histones H2A and H2B were found, by two independent labeling experiments, to be cross-linked to the DNA. Based on previous finding that the UV irradiation specifically cross-links residues which are in close proximity, irrespective of the nature of the amino acid side chain or the nucleotide involved, our results indicate that the four core histones are not positioned equivalently with respect to the DNA. This arrangement allows histones H2A and H2B to preferentially cross-link to the DNA. A water soluble covalent complex of DNA and histones was isolated. This complex was partially resistant to mild nuclease digestion, it exhibited a CD spectrum similar to that of chromatin, and was found to contain histone H1. These results are compatible with a model which suggests that histone H1, though anchored to the linker, is bound to the DNA at additional sites. By doing so it spans the whole length of the nucleosome and clamps together the DNA fold around the histone core.     

Buoyant density centrifugation of sea urchin embryo chromatin on sucrose-glucose gradient

Unsheard chromatin isolated from sea urchin embryos was submitted to buoyant density centrifugation in sucrose-glucose gradients. The main peak of blastula chromatin was at a density position of 1.299±0.028±0.009 g ml^-1 whereas at gastrula stage a shift to a lower buoyant density position of (1.276±0.021±0.007 g ml^-1) was observed. Besides the main peak, a small band with a density of 1.18 g ml^-1 was noticed. The lighter fraction differed from the heavy one in a higher histone to DNA ratio, a lower proportion of the F-1 histone, and a lower nonhistone to DNA ratio. The most pronounced developmental alterations of proteins were observed at the level of nonhistone protein patterns of the light fractions.     

Cell movements in the sea urchin embryo.

Recent studies show that gastrulation in the sea urchin embryo involves movement of cells over the blastopore lip (involution). Some cells in the vegetal plate of the late blastula become bottle-shaped but they play a limited role in gastrulation. The functions of specific integrins, regulators of cell-cell adhesion, and extracellular matrix components in gastrulation are currently being analyzed. In addition, light-microscopic studies continue to provide a unique picture of dynamic cell behavior in vivo.     

The identification by sequence homology of stage-specific sea urchin embryo histones H1

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I2, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B4 synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B4 itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B4 is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2',5'-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B4 synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B4 can limit their own activity in neutrophils.     

Stage-specific mRNAs coding for subtypes of H2A and H2B histones in the sea urchin embryo

Phagocytosis, pinocytosis and the surface distribution of concanavalin A (ConA) have been analyzed during mitosis in several mammalian cell lines. Use of the bisbenzimidazole dye, Hoechst 33258, for chromosome staining after gentle fixation made possible the rapid identification and correlation of mitotic phase with surface properties.Phagocytosis of both opsonized and nonopsonized particles is markedly depressed in mitotic cells of the mouse macrophage cell line J774.1. The uptake of opsonized particles (IgG-coated erythrocytes) is Impaired from early prophase through early G1, whereas phagocytosis of non-opsonized particles (latex beads) is restored by telophase. Fluid pinocytosis, determined by the uptake of soluble horseradish peroxidase, is also inhibited during mitosis. Thus peroxidase-containing cytoplasmic vesicles were virtually absent from mid-prophase through telophase in both J774 and Chinese hamster ovary (CHO) cells.Adsorptive pinocytosis of ConA was determined from the different distributions of fluorescence in single cells incubated at 37°C with rhodamine-conjugated ConA (surface and cytoplasmic label), then fixed and further incubated with fluorescein-conjugated anti-ConA (surface only). The separate fluorescence of Hoechst, fluorescein and rhodamine could be optically isolated. In interphase J774 cells, ConA is rapidly internalized into cytoplasmic vesicles. In contrast, ConA is restricted to the plasma membrane from mid-prophase through telophase. In CHO, the depressed pattern of internalization is not fully established until metaphase.The surface distribution of ConA also varied dramatically as a function of mitotic phase. Between mid-prophase and early anaphase, the pattern of surface ConA-receptor complexes is diffuse. Once the cleavage furrow begins to develop, however, ConA moves into the region of the furrow. This was shown in J774, CHO and 3T3 mouse embryonic fibroblasts, and is probably universal. ConA movement into the membrane that overlies the microfilaments of the contractile ring is analogous to similar movements that occur in interphase cells during ConA cap formation and during the development of phagocytic pseudopods. The analogy emphasizes the common functional consequences of microfilament-membrane organization.It is evident that membrane processes which depend upon endocytosis-for example, certain hormone-induced signals-may be interrupted during mitosis. Inhibition of endocytosis thus may be a significant element in the control of cellular activities during mitosis and a strong influence on the properties of the emergent post-mitotic cell.     

Structural differences in the chromatin from compartmentalized cells of the sea urchin embryo: differential nuclease accessibility of micromere chromatin.

The chromatin structure of three cell types isolated from the 16-cell stage sea urchin embryo has been probed with micrococcal nuclease. In micromeres, the four small cells at the vegetal pole, the chromatin is found to be considerably more resistant to degradation by micrococcal nuclease than chromatin in the larger mesomere and macromere cells which undergo more cellular divisions and are committed to different developmental fates. The micromeres show an order of magnitude decrease in the initial digestion rate and a limit digest value which is one third that of the larger blastomeres; both observations are suggestive of the formation of a more condensed chromatin structure during the process of commitment, or as the rate of cell division decreases. The decreased sensitivity to nuclease for micromeres is similar to results reported for sperm and larval stages of development.     

Non-histone chromatin proteins from the sea urchin Strongylocentrotus droebachiensis sperm and embryo

Non-histone chromatin proteins (NHP) from sperm and gastrula of the sea urchin Strongylocentrotus droebachiensis have been studied. The results obtained show that the total amount of the NHP in the sperm chromatin is one-sixth of that in the gastrula chromatin. Certain notable differences in the electrophoretic banding patterns of the NHP have also been observed. SDS polyacrylamide gel electrophoresis of NHP revealed one major specific component of molecular weight 17000 in the sperm chromatin and three major specific fractions with molecular weights 14000; 15000 and 35000 in gastrula chromatin. Furthermore, the gastrula chromatin NHP contains about ten minor specific fractions in the molecular weight range 25 000 to 65 000. The relevance of these results to the control of gene activity is discussed.     

Automated sequential affinity chromatography of sea urchin embryo DNA binding proteins.

An automated method of running a tandem sequence of oligonucleotide affinity columns was used to purify factors that interact specifically with cis-regulatory sites of the CyIIIa cytoskeletal actin gene of the sea urchin embryo (Strongylocentrotus purpuratus). The method allows quantitative enrichment in a single chromatographic run of up to 12 different sequence-specific DNA binding proteins, each of which may then be readily purified to homogeneity by methods such as preparative gel electrophoresis. The affinity chromatography and identification of six different CyIIIa-regulatory factors is described, and the general utility of the method is discussed.     

Isolation and properties of the initiation of DNA replication in the sea urchin embryo

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA from embryos of the sea urchin Strongylocentrotus intermedius, extruded DNA enriched for replication origins was obtained. The average length of the fragments of the DNA of this fraction was estimated to be about 800 base pairs. The origin-enriched nascent DNA strands were assayed for the presence of inverted repeats. The results show that the origin-enriched DNA is also enriched in inverted repeats. The bulk of palindromes in the total nuclear DNA was estimated to be 200 base pairs in length and from the origin-enriched DNA-150 base pairs.     

Fibronectin in the developing sea urchin embryo

The presence of fibronectin in developing sea urchin embryos was studied uing immunofluorescence staining. The fluorescence pattern indicates that fibronectin is found on the cell surfaces and between cells in the blastula and gastrula stages, indicating that it plays a role in cell adhesion. Its presence on invaginating cells also suggests its involvement in morphogenesis during early development.