Search PubMed⌕ Search

Biomedical subjects

R Sperling

Publications and source records attributed to R Sperling.

At least 73 records · Page 4Linked to original sources
PubMed1979

Low resolution models of self-assembled histone fibers from X-ray diffraction studies.

X-ray diffraction data from self-assembled histone fibers are presented for three systems: H4, H3-H4, and the four core histones H2A, H2B, H3 and H4. These data have been obtained under conditions of high ionic strength and high protein concentration which are thought to promote histone conformation similar to that found in intact chromatin. The low angle equatorial scattering (R less than .05 A-1) is analysed, and, with additional constraints imposed by electron microscopy data, four low resolution fibrillar models are derived. Two features common to all the possible models are a maximum outer diameter of approximately 60 A and a subfibril diameter of approximately 25 A. It is the interference of the protein subfibrils across a central region of low electron density - a 10 A "hole" - which gives rise to the characteristic diffraction peak at 36 A. Possible relationships of the models of the histone fibers to the structure of the histone component of chromatin are suggested.

Histones↗
PubMed1978

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.

Animals↗
PubMed1977

Molecular homogeneity of the histone content of HeLa chromatin subunits.

Interaction of affinity chromatographically purified antihistone H3 and antihistone H4 with isolated HeLa core particles, followed by separation of unreacted and reacted particles by sedimentation, demonstrates that every core particle contains these histones. Taken together with our previous data indicating the presence of H2B in every nucleosome (Simpson, R. T., and Bustin, M. (1976), Biochemistry 15, 4305), these data lead to the conclusion that each core particle contains two each of the four smaller histones. In contrast to the lack of interference in binding of more than one molecule of antibody to a single species of histone to the core particle, steric hindrance exists when attempts are made to bind both anti-H3 and anti-H4 to core particles.

Antibodies↗
PubMed1977

Arrangement of subunits in assembled histone H4 fibers.

The structure of assembled histone H4 fibers has been studied by analysis of electronmicrographs, including optical diffraction. An individual fiber has the appearance of an 80-A wide ribbon, twisted at intervals of about 330 A. Thicker fibers which have been observed seem to be bundles of ribbons. In diffraction patterns from both kinds of fiber, layer lines at axial spacings of about 1/55 A-1, 1/37 A-1, and 1/27 A-1 were most consistently observed. The possible arrangements of molecules within the twisted ribbons have been deduced and are found to be fairly closely related. The ribbons appear to consist of two parallel, unstaggered rows of repeating units, which are probably H4 dimers. The similarity between the observed layer line spacings of the H4 fibers and the spacings of the maxima in x-ray diffraction patterns from whole chromatin suggests that the H4 fibers have a structure related to that of chromatin. Since homogeneous preparations of histones H2A, H2B, and H3, or any mixture of these four histones, can form similar structures, it seems likely that the basic organization of chromatin is determined by a fibrous histone core around which the DNA is wrapped.

Histones↗
PubMed1976

Chromatin structure visualization by immunoelectron microscopy.

Antibodies elicited in rabbits by chromatin and by purified histone H2B have been used to study the structure of chromatin by immunoelectron microscopy. Chromatin spread on grids reveals a structure of closely packed spherical particles with an average diameter of 104 A, arranged either in clusters or in linear arrays of beads, some of which have a supercoil-like arrangement. No DNA strings connecting the beads could be observed. Upon antibody binding, the diameter of the particles increases up to 300 A. This size is compatible with a model where one layer of gamma globulin molecules 110 A long encircles a sphere of chromatin 100 A in diameter. The presence of rabbit gamma globulins on the enlarged beads has been verified by the addition of ferritin-labeled goat anti-rabbit gamma globulins. Anti-chromatin sera which react with nonhistone proteins but not with free histones or DNA react with more than 95% of the beads; this suggests that most of the beads contain nonhistone proteins. Since the number of nonhistone proteins is large, it is improbable that each sphere contains a full complement of these proteins. We therefore suggest that the various chromatin spheres contain different types of nonhistone proteins. About 90% of the chromatin spheres reacted with antibodies to histone H2B, suggesting the most of the chromatin beads contain this type of histone.

Antigen-Antibody Reactions↗
PubMed1976

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.

Animals↗
PubMed1976

The origin of the chemiluminescence of phagocytosing granulocytes.

Granulocytes engaged in the phagocytosis of opsonized zymosan emit light by a process that is inhibited by superoxide dismutase and catalase. In the present report is is shown that light emission is the result of reactions between certain unspecified constituents of the ingested particles and some or all of the oxidizing agents (H2O2, O2),and possibly the hydroxyl radical and singlet oxygen) produced by the activated cells. This conclusion is based on a study of light emission by both activated cells ans artificial O2 generating system containing xanthine oxidase and purine. With these two systems light production required the presence of both zymosan and oxidizing agent, suggesting that the oxidation of particle components is necessary for luminescence to occur. The characteristics of the emission spectrum as well as the finding that granulocytes activated by a nonparticulate agent (F-) fail to liminesce show that light emission by the relaxation of singlet oxygen to the ground state does not contribute in a major way to the chemiluminescence of phagocytosing granulocytes; whether singlet oxygen contributes to chemiluminescence in other ways cannot be decided from the data available. Inasmuch as the oxidation of constituents of ingested particles is an important bacterial killing mechanism in the granulocyte, chemiluminescence may be viewed as a manifestation of the microbicidal activity of the cell.

Catalase↗
PubMed1975

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.

Animals↗