The role of H2B histones from the sea urchin sperm in the formation of supranucleosome structures

The structural role of histone H2B from sea urchin sperm (H2Bsp) has been examined in experiments on reconstitution of chromatin from DNA and core histones taken in three variants: (1) four core histones from sea urchin sperm; (2) four core histones from calf thymus; (3) (H3, H4, H2A) from calf thymus and H2Bsp. It is shown that H2Bsp when present in reconstituted chromatin induces its aggregation. Fidelity of the reconstitution of nucleosomes has been tested using DNase I probe, one- and two-dimensional electrophoresis and electron microscopy. The reconstitutes that contain H2Bsp appear under electron microscope mainly as regular closely spaced large granules, about 450 A in diameter, which are very similar to the granules found in "native" sea urchin sperm chromatin. The reconstitutes formed by four core histones from calf thymus appear as randomly arranged particles, about 100 A in diameter. We conclude that histone H2Bsp participates in interactions between nucleosomes and is involved in the formation of the condensed supranucleosomal structure in sea urchin sperm chromatin.     

A histone H2B variant from the embryo of the sea urchin Parenchinus angulosus

A variant of histone H2B has been isolated from sea urchin embryo (Parenchinus angulosus). Out of the 53 amino acids positioned in the three CNBr-peptides only 26 residues are identical to those in the corresponding positions of calf thymus histone H2B. A similar degree of homology exists between the embryonic variant and the previously characterized variants from sperm cells of the same organism.     

Processes of reconstruction and association by nucleosomes with various formations of histones

The experiments on reconstruction of chromatin (without H1) from DNA and histone octamer containing either H2B from sea urchin sperm (H2B-S) or H2B from calf thymus are reported. It has been shown that H2B-S affects the mode of interaction of histones with DNA during the reconstitution of nucleosomal particles on one hand and on the other hand H2B-S plays a major role in the interactions of reconstituted mononucleosomes. These interactions result in supranucleosomal structures.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Nucleosomal structure of sea urchin and starfish sperm chromatin. Histone H2B is possibly involved in determining the length of linker DNA.

Comparison has been made between sea urchin and starfish sperm chromatin. The only protein by which chromatins from these sources differ significantly is histone H2B. Sea urchin sperm H2B is known to contain an elongated N-terminal region enriched in Arg. Analysis of the micrococcal nuclease digests of sea urchin and starfish nuclei in one- and two-dimensional electrophoresis has shown that sperm chromatin of both animals consists of repeated units similar in general features to those of rat thymus or liver. However, DNA repeat length in chromatin of sea urchin sperm (237 bp) is higher than that of starfish sperm (224 bp), while the core DNA length does not differ and is the same as in the chromatin of rat liver or thymus. A suggestion has been made that the N-terminal region of histone H2B is associated with the linker DNA and is responsible for the increased length of sea urchin linker DNA.     

A study of histones in amoeba nuclei by indirect immunofluorescence method.

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

The role of histone H2B from sea urchin sperm in the association of reconstituted minichromosomes

A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.     

Electron spin resonance studies on the conformation and interactions of histories containing cysteine. Histones H3 from calf thymus and H4 from sea urchin sperm.

In this study the spin-label method has been used to obtain information about conformational properties of regions containing cysteine of histone H3 from calf thymus, histone H4 from sperm of the sea urchin Arbacia lixula, and the histone complex H3–H4. It has been found that the microenvironments of histone H3 causing immobilization of the spin labels are sensitive to variations in ionic strength of dilute solutions of phosphate buffer, are partially destroyed by urea, and fully destroyed by proteolytic enzymes. The interaction of spin-labeled histone H3 with histone H4 induces an increase of immobilization of the spin label, indicating an increase in rigidity at the cysteine region of histone H3. The use of a series of spin labels of variable length for histone H3 gives an estimate of 0.8–1.0 nm for the apparent depth of the spin label binding site, a value which does not change upon interaction of histone H3 with H4. Histone H4 from A. lixula sperm causes a similar immobilization of the spin label. As for histone H3, immobilization increases with the ionic strength, and the structures are destroyed by urea and proteolytic enzymes. Upon mixing with histone H3, however, the extent of immobilization appears only slightly changed, and together with sedimentation velocity results, these studies suggest that the spin label attached to histone H4 prevents the complex formation.     

Phenylalanine metabolism in isolated rat liver cells. Effects of glucagon and diabetes.

Tryptic digestion of histone H1 from the sperm of the sea urchin Sphaerechinus granularis leaves a limiting peptide of approx. 80 residues that is of similar size to the limit peptide from calf thymus H1 or chicken erythrocyte H5. The S. granularis limit peptide folds to form tertiary structure similar to that of the intact parent histone H1 (shown by n.m.r. spectra), but the helical content is decreased by the digestion from 64 residues to 28. In contrast, intact calf thymus H1 and chicken erythrocyte H5 histones have only about 28 helical residues, which are preserved in their limit peptides. The extra helix in S. granularis is shown to be rapidly digested away by trypsin, and its location in histone H1 is discussed. A possible relationship of this structural feature to the length of linker DNA is proposed.     

The histones of the sperm of Spisula solidissima include a novel, cysteine-containing H-1 histone

The histones remaining at the end of the spermiogenic differentiation, which are found associated with a highly basic protamine-like component [Ausio, J. and K.E. Van Holde (1987) Eur. J. Biochem. 165, 363-371] in the mature sperm of Spisula solidissima, have been isolated and characterized for the first time. All four core histones H2A, H2B, H3, H4, and the lysine-rich histone H1 are present. The core histones are found in equal stoichiometric amounts. As has been observed in other bivalve molluscs, the amino acid compositions of the core histones of S. solidissima sperm are very close to those of their counterparts in the calf thymus somatic histones. The spermatic histone H1 exhibits an amino acid composition and structural features similar to other histones of the histone H1 family. Yet this latter histone seems to be sperm-specific, and it contains at least two cysteine residues per molecule, which makes it unique in its class.     

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.     

Immunological analysis of histone H2A from the slime mold Physarum polycephalum

Specific antibodies against the histone H2A from calf thymus were generated by injecting rabbits with complexes: histone H2A-RNA with a protein to RNA ratio of 3:1. In the microcomplement fixation assay the antibodies against the histone H2A from calf thymus immuno-reacted with the histone H2A from calf thymus but not with H2A from Physarum polycephalum. The histone H2A from calf thymus therefore appears to have an immunological determinant(s) which does not exist in H2A from Physarum polycephalum.     

An electrophoretic comparison of the histones of various strains of Tetrahymena pyriformis

Histones were extracted from isolated macronuclei of several strains of the ciliated protozoan Tetrahymena pyriformis and compared by electrophoresis on both urea-acrylamide and sodium dodecyl sulfate-acrylamide gels. High resolution urea-acrylamide-gel electrophoresis resolves Tetrahymena histones into five main classes. The lysine-rich histone H1 exhibits microheterogeneity within each strain, mostly due to phosphorylation, and varies extensively in electrophoretic mobility and apparent molecular weight among the strains. Both H3 and H4 are constant among Tetrahymena strains and consist of several secondarily modified subspecies. However, while the electrophoretic constancy of H4, observed in higher organisms, extends to this lower eukaryote, H3 of each Tetrahymena strain migrates faster than calf thymus H3 in both gel systems. This suggests that H3 is not as rigidly conserved as H4. Fraction HX has no electrophoretic counterpart in calf thymus histone. It consists of five subfractions, each of which displays a remarkably constant electrophoretic mobility among the various strains. H2B is electrophoretically variable among Tetrahymena strains. The intersyngen and interphenoset diversity of Tetrahymena histone is shown to be comparable to that found among vertebrate classes.     

A study on histones in amoeba nuclei by indirect immunofluorescence method

Nuclear proteins of four species of free-living Amoebidae (Amoeba proteus, A. discoides, Chaos carolinensis and Polychaos dubia) have been studied by indirect immunofluorescence technique using specific antisera to H1, H2A, H2B, H3 and H4 histone fractions from the calf thymus. It has been shown that the nuclei of the species examined have all these five histone fractions. However, the degree of similarity between homologous fractions from amoebae and the calf thymus varies and can be expressed in terms of immunological distance. Immunological differences between amoebic and calf thymus histones are the most pronounced in H1, being least in H3 and H4. Judged by its immunochemical characteristics, the histone fraction H2A from P. dubia is closer to the corresponding fraction from the calf thymus than is H2A from the other three amoeba species.     

S(T)PXX motifs promote the interaction between the extended N-terminal tails of histone H2B with "linker" DNA.

The assembly of hybrid core particles onto long chicken DNA with histone H2B in the chicken histone octamer replaced with either wheat histone H2B(2) or sea urchin sperm histone H2B(1) or H2B(2) is described. All these histone H2B variants have N-terminal extensions of between 18 and 20 amino acids, although only those from sea urchin sperm have S(T)PXX motifs present. Whereas chicken histone octamers protected 167 base pairs (bp) (representing two full turns) of DNA against micrococcal nuclease digestion (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813), all the hybrid histone octamers protected an additional 17-bp DNA against nuclease digestion. This protection was more marked in the case of hybrid octamers containing sea urchin sperm histone H2B variants and similar to that described previously (Lindsey, G. G., Orgeig, S., Thompson, P., Davies, N., and Maeder, D. L. (1991) J. Mol. Biol. 218, 805-813) for hybrid histone octamers containing wheat histone H2A variants all of which also have S(T)PXX motifs present. Continued micrococcal nuclease digestion reduced the length of DNA associated with the core particle via 172-, 162-, and 152-bp intermediates until the 146-bp core particle was obtained. These DNA lengths were approximately 5 bp or half a helical turn longer than those reported previously for stripped chicken chromatin and for core particles containing histone octamers reconstituted using "normal" length histone H2B variants. This protection pattern was also found in stripped sea urchin sperm chromatin, demonstrating that the assembly/digestion methodology reflects the in vivo situation. The interaction between the N-terminal histone H2B extension and DNA of the "linker" region was confirmed by demonstrating that stripped sea urchin sperm chromatin precipitated between 120 and 500 mM NaCl in a manner analogous to unstripped chromatin whereas stripped chicken chromatin did not. Tryptic digestion to remove all the histone tails abolished this precipitation as well as the protection of DNA outside of the 167-bp core particle against nuclease digestion.     

Homology of histones H2B from calf thymus and P4B from slime mold Physarum polycephalum

A comparative study of the amino acid composition of histone fractions P4b from slime mold Physarum polycephalum and H2B from calf thymus was carried out using peptide mapping. It was shown that 75% of peptides are common for both proteins. The slime mold histones contain two fractions (P4B and P3), which are homologous to the H2B histone fraction of calf thymus. The data of amino acid analysis, peptide mapping and some physico-chemical properties of the histones revealed the following correlation of the two types of histone fractions: P1--H1, P4a--H3, P4b and P3--H2B, P5-H2A, P6--H4.     

Histones of the fungi, Endomyces magnusii and Neurospora crassa]

Histones of Endomyces magnusii and Neurospora crassa were found to consist of four main fractions similar to calf thymus histones in their electrophoretic mobilities, molecular sizes and chromatographic behaviour on Akrilex P-60. Two of them are homologous to the most conservative histones H3 and H4. Other two fractions correspond to the histones H2A and H2B; however, they have some pecularities. A fraction of N. crassa histones corresponding to the H2B was isolated in a homogeneous state by means of gel filtration. It appeared to be very similar to calf thymus histone H2B in its amino acid composition.     

Antigenic variability among very-lysine-rich histones from calf thymus and from sperm of different echinoderms

1. Very-lysine-rich histone of a mammal (calf thymus), two sea urchins (Arbacia lixula and Paracentrotus lividus) and a sea cucumber (Holothuria tubulosa) were compared by using immunological techniques. 2. The degree of antigenic variability among all these histones was measured by quantitative microcomplement fixation. An evoluntionary diagram for this group of histones has been established. 3. The histone from sea cucumber is placed in an intermediate situation between those for calf thymus and sea urchins, and the immunological distance between the histones of the two urchins is very short.     

Analysis of Histones from the Endosymbiont Nucleus of a Binucleate Dinoflagellate1,2

Histones were prepared from chromatin of the eukaryotic (endosymbiont) nucleus of Peridinium balticum (Levander) Lemmermann. The amino acid composition of whole histone was rich in lysine and similar to that of Olisthodiscus luteus and Euglena gracilis. Electropheretic analysis of these proteins in acidic-urea disc gels revealed four major bands: one with a mobility slightly lower than that of calf thymus HI; and three others which comigrated with calf H2B, H2A, and H4, respectively. The low mobility band was soluble in 5% perchloric acid and was sensitive to FeCl₃ destaining. Electrophoresis in slab gels containing 0.1% SDS revealed five major components, with approximate molecular weights of 23,000, 20,000, 15,000, 13,000, and 11,000, respectively. The 15,000 and 11,000 dalton histones had mobilities identical to those of calf H3 and H4, respectively. The two highest molecular weight components were soluble in 5% perchloric acid. No bands were found to comigrate with calf H2A or H2B but a band was present that migrated to a position intermediate between calf H2A and H4 (13,000 dalton histone). Two-dimensional gels consisting of acidic-urea gels in the first dimension and SDS gels in the second dimension revealed that the 20,000 dalton component and the 13,000 dalton component are not resolved in the acidic-urea gel. As a working hypothesis, it is suggested that two of the five bands seen in SDS gels represent an H1-like doublet, and two are analagous to H3 and H4, respectively. The remaining histone may replace H2A and H2B.     

Histone phosphorylation during sea urchin development

Studies on histone phosphorylation during transitions in chromatin structure occurringin vivoduring spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extractsin vitroand evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structurein vivoand provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.