Transparency in the eye region of an ostracod carapace ( Macrocypridina castanea, Myodocopida).

Many myodocopid ostracods are unusual in that they have well-developed compound eyes yet must view their environment through a shell. The cypridinid Macrocypridina castanea is relatively large among ostracods (about 5-10 mm) and is a pelagic predator. This species possess highly pigmented shells with a transparent region lying just above the eye. Here we examine the ultrastructure and transparency of this window using electron microscopy, serial-block face scanning electron microscopy and X-ray diffraction analysis and optical modelling. An internal, laminar stack was identified within the window region of the shell that formed a more regular half-wave reflector than in non-window regions, and where the distance between molecules in the chitin-protein fibrils decreases as compared to the non-window area. This results in excellent transmission properties-at around 99% transmission-for wavelengths between 350 and 630 nm due to its half-wave reflector organization. Therefore, blue light, common in the mid and deep sea, where this species inhabits, would be near-optimally transmitted as a consequence of the sub-micrometre structuring of the shell, thus optimizing the ostracod's vision. Further, pore canals were identified in the shell that may secrete substances to prevent microbial growth, and subsequently maintain transparency, on the shell surface. This article is part of the theme issue 'Bioinspired materials and surfaces for green science and technology'.

Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

Fluorescently labelled histones as probes of nucleosome structure. Preparation and general properties of methionine-labelled histone H4.

A fluorescent derivative of calf thymus histone H4 has been prepared by the reaction of methionine-84 with N-(iodoacetylaminoethyl)8-naphthylamine-1-sulfonic acid at pH 2.4 in 8 M urea. The preparation and characterization of this labelled histone is described. Fluorescence emission measurements indicate that the label on H4 undergoes a 3--5-fold increase in emission intensity when H4 self-interacts or binds to DNA alone or is incorporated in a synthetic nucleosome. The changes observed are consistent with the formation of varied apolar environments around methionine-84, due most likely to histone-histone rather than histone-DNA interactions. Preliminary experiments indicate that the precise emission intensity of labelled H4 in the nucleosome is quite sensitive to conditions of ionic strength and histone integrity.

A thermal denaturation study of chromatin and nuclease-produced chromatin fragments.

Calf thymus chromatin and nuclease-produced chromatin fragments have been examined by thermal denaturation measurements. Native chromatin gave a series of distinct melting transitions at 64, 73,79, and 85 degrees C in 0.25 mM EDTA pH8. Treatments such as dialysis, mechanical shearing, or sulfhydryl oxidation of histone H3 carried out on native chromatin significantly altered the derivative melting profiles by blurring the distinct transitions and shifting the highest melting transition to a lower temperature. Derivative melting profiles for electrophoretically purified chromatin fragments, monomer through hexamer, all resembled that obtained from dialyzed chromatin. These results suggest that higher order structures exist in chromatin that are easily disrupted. Since the products of micrococcal nuclease (EC3.1.4.7) digestion of the altered chromatins did not exhibit any major electrophoretic differences from those obtained from nuclei, than most likely the primary arrangements of histones along the DNA are the main determinant for cleavage sites.

Histone-histone interactions. I. An electrophoretic study.

Whole histone extracted from chromatin by either acid or protamine displacement was found by gel electrophoresis at pH7 to contain only two histone complexes, H2A-H2B and H3-H4, and uncomplexed histone H1. Although both complexes are dissociated at low pH or with high urea concentrations, removal of the denaturant resulted in complete complex reformation within minutes at the most. A syntematic investigation of binary, ternary and quaternary histone mixtures revealed that interactions also occur between histones H2B-H4 and H2A-H4. No evidence however was found for the formation of ternary and quaternary histone complexes.

Histone-histone interactions. II. Structural stability of the histone H3-H4 complex.

The stability of the histone H3-H4 complex toward urea, changes in pH and ionic strength, and certain chemical modifications have been examined by gel electrophoresis anc circular dichronism. When uncomplexed, the two cysteine residues of histone H3 become rapidly oxidized, forming an intramolecular disulfide bridge which apparently blocks complex formation on return to complexing conditions. The complex was found to be unstable toward low values of pH and ionic strength, concentrations of urea exceeding 1 M, modifications of the cysteine residues, and fragmention in which the C terminal portions of either H3 or H4 are removed. A possible structure for this complex is proposed.

Effect of histone H3 sulfhydryl modifications on histone-histone interactions and nucleosome formation and structure.

The effect of histone H3 sulfhydryl mnodification and disulfide bridge formation on histone-histone interactions, nucleosome reconstitution and structure has been examined for calf and chicken mononucleosomes. For intramolecular disulfide bridge formation histone H3-H4 complexation is disrupted and no nucleosome-like particle containing all four of the histones could be prepared. Intermolecular disulfide bridge formation between H3 residues 110 and 110 as well as chemical modification of this site with small and with bulky groups allowed histone H3-H4 complexation and the reconstituatioin of a nucleosome-like particle. However, the yield of such particles is decreased and their thermal denaturation properties indicate a reduced stability. These results suggest that the histone core is destabilized or even structurally altered by even a minor modification at H3 position 110, such as carboxymethylation, and therefore this site must be used with caution for the attachment of reporter groups.

DNA and histone synthesis in mouse cells which exhibit temperature-sensitive DNA synthesis.

It is demonstrated that temperature inactivation of histone synthesis is coupled to inhibition of DNA replication in ts AlS9 and ts Cl mouse L-cells, which are temperature-sensitive (ts) in an S-phase function. In contrast, uncoupling of histone and DNA synthesis occurs in BalB/C-3T3 ts 2 cells which are ts in a function of the pre-DNA-synthetic phase. Termination of histone synthesis in ts AlS9 and ts Cl cells is 16--18 h after onset of temperature inactivation of DNA replication and appears to be associated with general cessation of chromatin replication triggered by the earlier event. Synthesis of histone and other chromosomal proteins proceeds in ts 2 cells under conditions in which DNA synthesis undergoes temperature inactivation. It is suggested that the terminal phenotype of coupled temperature inactivation of DNA and histone synthesis may be diagnostic of cells ts in an S-phase function and may therefore be a useful secondary screen in designation of cell cycle mutants.

Conformational studies on histone H3 and its CNBr peptides.

Histone H3 and H3 peptides 1--120, 1-90, 91-135, 91-120 and 121-135 have been prepared and examined for salt-induced conformational changes by circular dichroism measurements. It was found that reduced histone H3 and the reduced peptides 1-120, 91-135 and 91-120 exhibit biphasic changes with the formation of alpha-helix and beta structures. H3 peptide 1-90, on increasing the ionic strength to moderately high levels, monophasically formed appreciable quantities of alpha-helix and beta structures, while peptide 121-135 remained unfolded under all ionic strengths examined. All the above peptides except 121-135 also aggregate when the ionic strength is raised. The salt-induced near-ultraviolet circular dichroic spectra of histone H3 and peptide 1-90 were found to be very similar, suggesting that the conformational changes induced in the peptide 1-90 are essentially the same as those observed for the intact histone. These results support the contention that the polypeptide segments of this histone interact initially by parallel self-association followed by the formation of even larger aggregates on a longer scale.

Purification and characterization of two porcine liver nuclear histone acetyltransferases.

Two forms of porcine histone acetyltransferase (types I and II) have been purified to apparent homogeneity from liver nuclei. Both activities are extracted from nuclei by 0.5 M NaCl and display a native Mr of 110,000 as determined by gel filtration. Saline enzyme extracts were subject to ammonium sulfate precipitation and sequential chromatography on Q-Sepharose, Sephacryl S-200, hydroxylapatite, and Mono Q supports. The histone acetyltransferase type I fraction contains three polypeptide chains with apparent Mr values of 105,000, 62,000, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide peptide mapping and immunoblotting suggest that the Mr 62,000 and 45,000 polypeptides are derived by cleavage of the Mr 105,000 polypeptide. Histone acetyltransferase type II contains two different subunits with apparent Mr values of 50,000 and 40,000, respectively. The amino acid composition, heat inactivation profiles, and Michaelis constants with respect to both acetyl coenzyme A and histones were indistinguishable for types I and II. However, affinity-purified polyclonal antibodies to both forms of the enzyme do not cross-react; cyanogen bromide-derived in situ cleavage digest patterns show few similarities; and the turnover number for type I is approximately 15-fold lower than that for type II. We estimate that there is one enzyme molecule for every 500 nucleosomes. The existence of two distinct forms of nuclear histone acetyltransferase in pig liver suggests that they may have separate functions in vivo.

Intermolecular histone H4 interactions in core nucleosomes.

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.

Conformations of the core nucleosome: effects of ionic strength and high mobility group protein 14 and 17 binding on the fluorescence emission and polarization of dansylated methionine-84 of histone H4.

Chicken histone H4 labeled at Met-84 with the fluor N-[(acetylamino)ethyl]-8-naphthyl-amine-1-sulfonic acid has been incorporated into a nucleosome which has physical characteristics virtually identical with those of native core nucleosomes. The fluorescence emission and polarization properties of the labeled nucleosome were measured as a function of ionic strength and the binding of high mobility group (HMG) proteins 14 and 17. Also, the accessibility of the fluor to the quenching agent acrylamide was determined. It was found that the fluorescence emission changes in the range 0.1-1000 mM NaCl are rather small and indicate that no major unfolding of the octamer structure occurs around Met-84 on H4 at least. Five or perhaps six discrete states were found in that ionic strength range. Each has a different accessibility to the quenching agent. The range of accessibilities varied from 9 X 10(-7) to 32 X 10(-7) mol-1 s-1 for 0.1-1000 mM NaCl, respectively. Polarization measurements showed that there was little change in the rotational relaxation lifetime of the fluor at ionic strengths less than 50 mM NaCl. Above this value, the rotational relaxation lifetimes decreased from 107 to 25 ns at 600 mM NaCl, indicating a moderately increased rotational freedom for the fluor. It is suggested that the histone octamer changes its degree of compaction in the range 0.1-600 mM NaCl but that no major protein unfolding occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Internal architecture of the core nucleosome: fluorescence energy transfer studies at methionine-84 of histone H4.

Chicken histone H4, labeled separately at Met-84 with N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid and 5-(iodoacetamido)fluorescein, was reassociated with unlabeled histones H2A, H2B, and H3 and 146 base pairs of DNA to produce fluorescently labeled nucleosomes having physical characteristics virtually the same as those of native core particles. Four types of particles were prepared containing respectively unlabeled H4, dansylated H4, fluoresceinated H4, and a mixture of the two labeled H4 molecules. Quantitative singlet-singlet energy-transfer measurements were carried out to determine changes in the distance between the two Met-84 H4 sites within the same nucleosome following conformational transitions which we have reported earlier. In the ionic strength range 0.1-100 mM NaCl, the distance between these sites is less than 2 nm except at 1 mM. Between 100 and 600 mM monovalent salt the distance separating the donor and acceptor fluors at Met-84 H4 increases to 3.8 nm. The conformational change centered around 200 mM NaCl is cooperative. Our results and those of others indicate that there is little unfolding of the histone octamer, at least around Met-84 H4, in the entire ionic strength range studied. A mechanism involving the rotation of the globular portion of H4 is proposed to account for this transition which occurs at physiological ionic strengths.

A diffusible auxin from Pinus radiata pollen and its possible role in stimulating ovule development.

Germinated pollen of Pinus radiata contains an auxin which is active in the Avena coleoptile test. It differs from all other hormones detected in pine pollen in that it is readily able to diffuse out from the pollen into an agar medium. It is suggested that, following pollination in vivo, the auxin may diffuse from the germinated pollen-tube onto the nucellus, thereby triggering the processes which allow ovule and gametophyte development to proceed. The auxin is water-soluble and may be an indole derivative.

Histone H3 thiol reactivity and acetyltransferases in chicken erythrocyte nuclei.

Chicken erythrocyte nuclei previously incubated separately with two novel mercury compounds (N-chloromercuribenzoyl)-biocytin and bis(p-(chloromercuribenzoyl))-[3H]lysine diamide) were digested with micrococcal nuclease and the digest products fractionated according to their solubility in 0.15 M NaCl and molecular size. The identity and quantitation of the chromatin fractions and proteins containing covalently bound mercury were determined by Western blotting, autoradiography, and scintillation counting. The most highly acetylated species of histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction also contained the highest proportion of bound mercury. This fraction contains hyperacetylated core histones, is depleted in linker histones, and enriched in nonhistone proteins. Histone H3 in the 0.15 M NaCl-soluble mononucleosomes, which are unacetylated and lack linker histones, was 45% less labeled than histone H3 in the 0.15 M NaCl-soluble polynucleosome fraction. In the 0.15 M NaCl-insoluble polynucleosomes, which contain unacetylated histones and molar proportions of linker histones, histone H3 was 63% less labeled. Allowing for the differential abundance of these subfractions in the nucleus, the relative H3 reactivities are 50, 7, and 1 for 0.15 M NaCl-soluble polynucleosomes, mononucleosomes, and 0.15 M NaCl-insoluble polynucleosomes, respectively. Thus a gradation of reactivities exists which correlates with increasing hyperacetylation and linker histone depletion. High mobility group proteins 1 and 2, found in subnucleosome particles in the 0.15 M NaCl-soluble fraction, are extensively mercury-labeled. Distribution of histone acetyltransferase activity among salt- and size-resolved micrococcal nuclease produced fractions was almost 5-fold greater in the 0.15 M NaCl-soluble supernatant than in the 0.15 M NaCl-insoluble pellet. Furthermore, the acetyltransferase activity, which is tightly bound to undigested chromatin, is rapidly released by both micrococcal nuclease and DNase I. For short digestion times the enzyme is associated with the salt-soluble polynucleosomes, but at longer times of digestion the enzyme appears to be free from intact nucleosomes. The enzyme may be localized in the globin domain in erythrocytes and maintains that region in a hyperacetylated state which results in an altered linker histone binding reflected in a change in the reactivity of the usually inaccessible H3 cysteine 110.

Histone accessibility determined by lysine-specific acetylation in chicken erythrocyte nuclei.

N-Hydroxysulfosuccinimidyl [3H]acetate was synthesized and, following the determination of the optimal reaction conditions, was used to acetylate histones in chicken erythrocyte nuclei at 4 degrees C, pH 8. The histones were extracted from the labelled nuclei and the distribution of the acetyl groups determined from the amount of tritiated acetate in isolated peptides. The relative degree of acetylation of molecules was H1 1.0, H5 0.81, H2B 0.48, H2A 0.24, H3 0.24, H4 0.16. Histone H1 is the most exposed histone followed by H5. The core histones are much less accessible to chemical modification than the linker histones by a factor of 4-5. Histones H2A, H2B and H5 appear to be labelled at random along the entire polypeptide chain, while histones H3 and H4 are labelled almost exclusively in the first 30 residues from the N terminus. Control and acetylated chicken erythrocyte nuclei were digested with DNase I and the resulting DNA hybridized to globin and ovalbumin cDNAs. Acetylation, at 14 molecules acetate/core nucleosome or 20 molecules acetate/chromatosome, increased the DNase I sensitivity of the ovalbumin gene to that of the globin sequences in the control sample, while the globin sequences became even more nuclease-sensitive. Our results suggest that increased sensitivity of chromatin towards nuclease digestion might be due to increased solubility of the chromatin fibre.

Histone-induced damage of a mammalian epithelium: the role of protein and membrane structure.

In a previous report [T. J. Kleine, A. Gladfelter, P. N. Lewis, and S. A. Lewis, Am. J. Physiol. 268 (Cell Physiol. 37): C1114-C1125, 1995], we found that the cationic DNA-binding proteins histones H4, H1, and H5 caused a voltage-dependent increase in the transepithelial conductance in rabbit urinary bladder epithelium. In this study, results from lipid bilayer experiments suggest that histones H5-H1 and H4 form variably sized conductive units. Purified fragments of histones H4 and H5 were used to determine the role of histone tertiary structure in inducing conductance. Isolated COOH- and NH2-terminal tails of histone H4, which are random coils, were inactive, whereas the central alpha-helical domain induced a conductance increase. Although the activities of the central fragment and intact histone H4 were in many ways similar, the dose-response relationships suggest that the isolated central domain was much less potent than intact histone H4. This suggests than the NH2- and COOH-terminal tails are also important for histone H4 activity. For histone H5, the isolated globular central domain was inactive. Thus the random-coil NH2- and COOH-terminal tails are important for H5 activity as well. These results indicate that histone molecules interact directly with membrane phospholipids to form a channel and that protein tertiary structure and the degree of positive charge play an important role in this activity.

Rearrangements and reconstitution of mononucleosomes as monitored by thermal denaturation measurements.

Derivative melting profiles of calf thymus mononucleosomes have been examined for changes resulting from variations in solvent pH and ionic strength, histone H1 content, and DNA size. Samples of mononucleosomes were found to rearrange during freeze-drying to form an altered monomer and a series of noncovalent multimers. The derivative melting profiles of these particles differ significantly from those for the untreated monomer and dimer. The noncovalent dimer exhibited a new melting transition at 66 degrees C involving approximately 18 base pairs of DNA normally associated with the highest melting transition. Mononucleosomes were reconstituted from 6 M guanidine hydrochloride to give particles with physical properties including melting profile which were virtually indistinguishable from those of the starting material. This result confirms the notion that no structural domains exist in the histone core that can be irreversible denatured by noncovalent perturbations.