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D E Olins

Publications and source records attributed to D E Olins.

At least 55 records · Page 3Linked to original sources
PubMed1983

Neutron scattering studies of nucleosome structure at low ionic strength.

Ionic strength studies using homogeneous preparations of chicken erythrocyte nucleosomes containing either 146 or 175 base pairs of DNA show a single unfolding transition at about 1.5 mM ionic strength as determined by small-angle neutron scattering. The transition seen by some investigators at between 2.9 and 7.5 mM ionic strength is not observed by small-angle neutron scattering in either type of nucleosome particle. The two contrasts measured (H2O and D2O) indicate that only small conformational changes occur in the protein core, but the DNA is partially unfolded below the transition point. Patterson inversion of the data and analysis of models indicate that the DNA in both types of particle is unwinding from the ends, leaving about one turn of supercoiled DNA bound to the histone core in approximately its normal (compact) conformation. The mechanism of unfolding appears to be similar for both types of particles and in both cases occurs at the same ionic strength. The unfolding observed for nucleosomes in this study is in definite disagreement with extended superhelical models for the DNA and also disagrees with models incorporating an unfolded histone core.

Animals↗
PubMed1983

Electron microscope tomography: transcription in three dimensions.

Three-dimensional reconstruction of an asymmetric biological ultrastructure has been achieved by tomographic analysis of electron micrographs of sections tilted on a goniometer specimen stage. Aligned micrographs could be displayed as red-green three-dimensional movies. The techniques have been applied to portions of in situ transcription units of a Balbiani ring in the polytene chromosomes of the midge Chironomus tentans. Current data suggest a DNA compaction of about 8 to 1 in a transcription unit. Nascent ribonucleoprotein granules display an imperfect sixfold helical arrangement around the chromatin axis.

Animals↗
PubMed1982

pH effects on the structure of the inner histones.

The structure of the inner histone complex extracted from chicken erythrocyte chromatin with 2 M NaCl has been studied as a function of pH. At pH 6, the complex dissociates to (H3-H4)2 tetramer and H2A.H2B dimer, with little change in alpha-helix content (as monitored by circular dichroism at 222 mm). Although the circular dichroism of tyrosyl side chains is also largely unchanged by the dissociation, measurements of intrinsic fluorescence do suggest a change in the environment of one or more tyrosines as a result of dissociation. Below pH 4, the histones become partially unfolded, lose specific secondary and tertiary structure, and undergo nonspecific aggregation. Both the pH 6 and 4 transitions, which are largely reversible, parallel pH-induced structural changes of nucleosomes (Zama, M., Olins, D.E., Prescott, B. and Thomas, G.J. (1978) Nucleic Acids Res. 5, 3881-3897). The results are consistent with the presence of tyrosine residues at the histone subunit-subunit contacts and suggest that histone conformation within the globular regions is largely independent of histone-DNA interactions.

Animals↗
PubMed1982

Neutron scattering studies and modeling of high mobility group 14 core nucleosome complex.

Considerable evidence relates the nonhistone proteins high mobility group (HMG) 14 and HMG 17 with the structure of active or potentially active chromatin. In this study, bulk nucleosome core particles prepared from chicken erythrocytes and the complex formed by binding two HMG 14 molecules per nucleosome core were studied by use of small-angle neutron scattering techniques. By varying the H2O/2H2O ratio, and hence the contrast between the solvent and the particles, it was possible to determine the radius of gyration of the protein and of the DNA independently and as a function of HMG 14 binding. The results show an increase of 0.9 +/- 0.6 A (mean +/- SEM) in the protein radius of gyration and of 2.7 +/- 0.6 A in the DNA radius of gyration upon binding of HMG 14 to the nucleosome. These changes are considered in the light of several postulated modes for the unfolding or perturbation of the nucleosome structure. Modeling calculations demonstrate that the observed changes in radius of gyration for the DNA and for the protein are too small to be consistent with an overall unfolding or opening of the core particle upon HMG 14 binding. However, the observed changes are consistent with several models that involve only minor changes in the structure. It is postulated that the differences observed may be an indication of the type of conformational change occurring in active nucleosomes.

Animals↗
PubMed1982

Ultrastructural studies of Chironomus salivary gland cells in different states of Balbiani ring activity.

Chironomus tentans fourth instar larvae were awakened from oligopause (diapause) and treated with pilocarpine to stimulate Balbiani ring transcriptional activity. Salivary glands were fixed, sectioned and examined by conventional and stereo-electron microscopy. Balbiani ring RNP lateral fiber densities were measured in well-formed regions with clear Balbiani ring granules arranged along chromatin transcription axes. Despite an up to ten-fold range in Balbiani ring transcriptional activity [10, 12, 14], we observed no significant difference in lateral fiber densities. These data are discussed in terms of the variety of mechanisms for modulating overall transcriptional activity, including recruitment of parallel transcription units in the polytene chromosome. The lack of other major ultrastructural changes at the nuclear envelope and within the domains of tubular rough endoplasmic reticulum argue strongly against a major dismantling of subcellular structure during the induced oligopause.

Animals↗
PubMed1982

Stereo electron microscope studies of mitotic chromosomes from Chinese hamster ovary cells.

Stereo electron microscopy was employed to examine thin sections of Chinese hamster ovary metaphase chromosomes in situ and of chromosomes released from Chinese hamster ovary cells by several methods. Detergent lysis of cells in a buffer containing Mg++ and Ca++ and hypotonic lysis of cells in a hexylene glycol-Ca++ buffer released chromosomes that exhibited a three-dimensional meshwork of about 50 nm chromatin fibers. Fragmentation of cells in serum-free medium by vortexing with glass beads revealed a more dispersed chromosomal morphology with a mesh of 10 to 25-nm fibers exhibiting a presumptive nucleosomal substructure. Possible origins of the various fiber sizes are discussed in terms of current models of metaphase chromosome structure.

Animals↗
PubMed1981

Stereo-electron microscopy of nuclear structure and replication in ciliated protozoa (Hypotricha).

Employing stereo-electron microscopy on sections (ca. 0.1 micrometer thick) of the ciliated protozoa Stylonychia, Oxytricha and Euplotes, we have examined the ultrastructure of macronuclear replication bands (RB) and micronuclei. The macronuclear RB is composed of two zones: a "forward zone" showing a special chromatin organization consisting of regular 40 to 50 nm diameter fibers with an indication of nucleosomal substructure; and a "rear zone", the site of DNA replication, consisting of a mesh of 10 nm chromatin fibers. Micronuclei exhibit chromatin strands of 60 to 70 nm diameter. Employing the Bernhard staining procedure, we have observed that the chromatin fibers of the RB and of the micronuclei remain unbleached, whereas macronuclear condensed chromatin is bleached of stain, indicating that the replication band and the micronuclei contain chromatin in a configuration different from that of other forms of densely packed chromatin. The regularity of the chromatin fibers within the forward zone is of particular interest since it is comparable to the regularity of chromatin seen in the transcriptionally-inactive chromatin of other nuclei such as avain erythrocytes and sea urchin spermatozoa. It is likely that the forward zone chromatin fibers consist of highly ordered arrangements of nucleosomes, associated with additional nonhistone proteins.

Animals↗
PubMed1980

Visualization of nucleosomes in thin sections by stereo electron microscopy.

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.

Animals↗
PubMed1980

Stereo-electron microscopy of nucleoli, Balbiani rings and endoplasmic reticulum in Chironomus salivary gland cells.

Employing stereo-electron microscopy on sections (ca. 0.1 micrometer thick) of isolated salivary glands from Chironomus tentans, we have formulated structural models for the organization of the peripheral nucleolar strands, the Balbiani ring (BR) transcription axes, and the tubular rough endoplasmic reticulum (RER). (i) The ca. 67 nm wide peripheral nucleolar strands display a higher-order organization of ca. 13.6 nm granules connected to a central axis, possibly representing a storage of pre-ribosomal RNP. (ii) The BR transcription axes were visualized in three-dimensions, and the density of lateral RNP fibrils was estimated (ca. 36 RNP fibrils/micrometer transcription axis). This estimate of the density of nascent RNP fibrils, suggests that in situ the DNA packing ratio of transcribing regions in intermediate between extended DNA and a ca. 10 nm polynucleosomal fiber. (iii) The tubular RER appears to reveal extended helical arrays of polyribosomes. The helices have an overall diameter of ca. 130 nm and exhibit a mean of 12 ribosomes per helical turn. We suggest that this arrangement of membrane-bound ribosomes represents a functional specialization of the cell, facilitating the translation of the very large BR mRNA of ca. 37 kb into a secretory product.

Animals↗
PubMed1979

The temperature and pH dependence of conformational transitions of the chromatin subunit.

Hydrodynamic, spectroscopic, and chemical crosslinking studies on monomer chromatin subnits are reported as a function of ionic strength, pH, and temperature. In earlier studies, two salt-dependent conformational transitions were described (Gordon et al., Proceedings of the National Academy of Science, 75, 660, 1978). Transition one occurred between 0.7 and 2.0 mM ionic strength and transition two occurred between 5.0 and 11.0 mM ionic strength. Crosslinking at 11 mM ionic strength with formaldehyde suppressed both transitions. In this communication we report that the second transition was characterized by changes in the circular dichroism spectra in the 260--320 nm region as well as by changes in the hydrodynamic properties. As the ionic strength was increased from 5.0 to 11.0 mM, [theta]282 decreased from 2000 TO 1500 DEG CM2/DMOLE AND [THETA]295 decreased from 0 to -400 deg cm2/dmole. Both transitions occurred in the pH range from pH 6.0 to 9.2. At pH 5.0, the two ionic strength-dependent transitions were no longer observed and the characteristic changes in the circular dichroism spectra were suppressed. The spectra of the monomer subunits at pH 5.0 showed only small changes with ionic strength and resembled the spectra of the subunits at 11 mM ionic strength above pH 6.0. In order to characterize the transitions in thermodynamic terms an ionic strength near the midpoint of each transition was selected. Then, changes in s20,w and D20,w were measured as a function of temperature. These data allow an estimation to be made of the enthalpies and entropies of the transitions.

Chromatin↗
PubMed1979

Interaction of benzo[alpha]pyrene diol-epoxide with nuclei and isolated chromatin.

Chicken erythrocyte chromatin and nuclei were labeled with benzo[alpha]-pyrene (B[alpha]P) diol-epoxide (anti) and digested with micrococcal nuclease to mono- and dinucleosomes. Analysis of the distribution of the carcinogen showed that the internucleosomal region bound 3-4 times more carcinogen per unit DNA than did nucleosomes. The enhanced binding of the 'ultimate' carcinogen to the internucleosomal region was similar when isolated chromatin or nuclei were used for in vitro labeling. Furthermore, isolation of the histone core proteins, H2A, H2B, H3 and H4, revealed that only 15% of the carcinogen was associated with the histones and that the majority of the carcinogen was bound to chromosomal DNA. Fluorography of purified nucleosomal histones showed that the covalent association of the carcinogen was mainly with histones H3 and H2B.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1979

Stereo electron microscopy of the 25-nm chromatin fibers in isolated nuclei.

Thin sections (0.1-0.25 micron) of isolated chicken erythrocyte nuclei were examined at various tilt angles. Stereo pairs of electron micrographs document the parallel alignment of 25-nm chromatin fibers adjacent to the nuclear envelope, and demonstrate a fiber substructure consistent with close-packed arrays of nucleosomes.

Animals↗
PubMed1979

Core nucleosomes by digestion of reconstructed histone-DNA complexes.

Reconstructed complexes of the inner histones (H2A, H2B, H3, H4) and a variety of DNAs were digested with micrococcal nuclease to yield very homogeneous populations of core nucleosomes (nu 1). Nucleosomes containing Micrococcus luteus DNA (72% G+C); chicken DNA (43% G+C), Clostridium perfringens DNA (29% G+C); or poly(A-dT.poly(dA-dT) have been examined by circular dichroism, thermaldetenaturation, electron microscopy, and DNAse I digestion. Circular dichroism spectra of all particles show a typically suppressed ellipticity at 260--280 nm and a prominent alpha-helix signal at 222 nm. All particles show biphasic melting except nu 1 (dA-dT), which show three prominent melting transitions at ionic strength less than or equal to 1 mM. DNAse I digestion of nu 1 (dA-dT) produces a ladder of DNA fragments fiffering in lengthy by one base residue. nu 1 (dA-dT) contain 146 base pairs of DNA and exhibit an average DNA helix pitch of 10.4-10.5 bases per turn. There appear to be two regions of different DNA pitch wihtin nu 1 (dA-dT). It is suggested that the two regions of DNA pitch might correspond to the two regions of the melting profiles.

Animals↗
PubMed1979

Salt-dependent interconversion of inner histone oligomers.

The inner histone complex, extracted from chicken erythrocyte chromatin in 2 M NaCL AT pH 7.4, has been characterized by sedimentation equilibrium and sedimentation velocity. High speed sedimentation equilibrium studies indicate that in 2 M NaCl the inner histones are a weakly associating system with contributions from species ranging in molecular weight from dimer to octamer. The appearance of a single boundary (3.8S at 2 M NaCl) in sedimentation velocity studies conducted over a wide range of protein concentrations and ionic conditions indicates that the various histone oligomers present are in rapid equilibrium with one another. At higher salts the equilibrium is shifted to favor higher molecular weight species; in 4 M NaCl essentially all of the histone is octameric at protein concentrations above 0.2 mg/ml. The facile interconversion of histone oligomers suggests that small alterations in histone-histone interactions may be responsible for changes in nucleosome conformations during various biological processes.

Animals↗
PubMed1979

Stereo-electron microscopy and energy-dispersive x-ray analysis of avian reticulocytes.

Utilizing the techniques of stereo-electron microscopy (stereo-EM) and energy-dispersive x-ray analysis (EDX), we have studied aspects of the ultrastructure of avian reticulocytes. Stereo-EM of thin sections (0.10 to 0.25 mum thick) stained with uranyl and lead revealed the three-dimensional arrangement of 25 nm chromatin fibers on the tangential surfaces of nuclei. Use of the Bernhard staining procedure in combination with stereo-EM permitted a three-dimensional view of the interchromatin spaces and channels leading to the nuclear pores, and of cytoplasmic polyribosomes. With either staining technique we frequently encountered in the cytoplasm clusters and paracrystalline arrays of electron-dense granules with granule diameters approximately 1/2 that of monomer ribosomes. These granules were highly electron-dense in unstained specimens and have been identified as intracellular ferritin on the basis of the similarity of their ultrastructural morphology to that of horse spleen ferritin and their high content of iron as determined by EDX. The possibility that these granules represent toxic products of phenylhydrazine treatment (Heinz bodies) is considered unlikely, since we have demonstrated in this study that Heinz bodies cannot be visualized in unstained preparations, do not reveal the same granular structure, and do not contain significant amounts of iron above background. The occurrence of intracellular ferritin is discussed in light of current concepts of iron transport and storage during erythropoiesis.

Anemia↗
PubMed1978

Conformational changes of the chromatin subunit.

Hydrodynamic studies on monomer chromatin subunits (v1) as a function of ionic strength (0.7 mM to 100 mM KCl) indicate two salt-dependent conformational transitions. An abrupt transition occurs at about 7.5 mM ionic strength. Decreasing the ionic strength from 10 to 5 mM results in a decrease in s20,w of the v1 from 11.1 to 9.9 S. The diffusion coefficient D20,w decreases from 3.3 to 2.7 X 10(-7) cm2 sec--1. The v1 crosslinked with formaldehyde at 10 mM ionic strength do not undergo a similar salt-dependent change in s20,w. Another transition is observed at about 1 mM ionic strength; s20,w decreases to 9.4 S and D20,w decreases to 2.2 X 10(-7) cm2 sec--1. Throughout the entire salt range the molecular weight of the v1 remains reasonably constant, implying that salt-dependent changes in the frictional coefficient are being observed. Various hydrodynamic models are considered as possible interpretations of the observed changes in the frictional coefficient.

Chromatin↗