Donald E. Olins

Spheroid Chromatin Units (ν Bodies)

Linear arrays of spherical chromatin particles (ν bodies) about 70 angstroms in diameter have been observed in preparations of isolated eukaryotic nuclei swollen in water, centrifuged onto carbon films, and positively or negatively stained. These bodies have been found in isolated rat thymus, rat liver, and chicken erythrocyte nuclei. Favorable views also reveal connecting strands about 15 angstroms wide between adjacent particles.

Model nucleohistones: The interaction of F1 and F2al histones with native T7 DNA☆

Abstract Synthetic complexes of the lysine-rich histone (F1) and of the glycine- and arginine-rich histone (F2al) with native T7 DNA have been prepared by the step-gradient dialysis procedure from 5 m -urea-3 m -NaCl and from 5 m -guanidine-HCl. Data on the physical properties of these complexes were obtained relevant to their solubility, thermal stability, sedimentation coefficients, circular dichroic spectra, and ultrastructure. Complexes of F1 with T7 DNA, prepared from urea-NaCl or from guanidine, exhibited a salt-dependent structural change between 10 −3 and 10 −1 m -sodium ions. At low ionic strengths, complexes sedimented like naked DNA and appeared to be a loose network of DNA-like fibers when examined in the electron microscope. At higher ionic strengths the complexes exhibited rapid and heterogeneous sedimentation and an altered circular dichroic spectrum, and they appeared in the electron microscope to be a mixture of large (0.1 to 0.3 μ diameter) donut and stem-like structures. Complexes of F2al with T7 DNA prepared from urea-NaCl and from guanidine revealed differences with respect to melting, sedimentation, circular dichroic, and electron microscopic properties. Complexes prepared from urea-NaCl sedimented as a single rapid heterogeneous component and resembled rosettes in the electron microscope; complexes from guanidine exhibited greater thermal stability, sedimented as two components, and revealed the presence of donut structures. Neither type of complex revealed salt-dependent structural changes of the magnitude seen with F1-DNA complexes. Poly- l -lysine and T7 DNA were combined by gradient dialysis from urea-NaCl, and also appeared as donuts and short stem structures over a wide range of ionic strengths. The present studies suggest that the structure of the annealed protein-DNA complexes strongly depends on the conformation of the basic protein during the initial stages of its interaction with native DNA. A highly disordered protein conformation results in characteristic donut and stem-like structures. F2al histone, however, aggregates in high NaCl concentrations, probably due to its hydrophobic portion (residues 46 to 102), resulting in rosette structures. The instability of F1-DNA donuts and stems at decreased ionic strength may result from the high proline content in the basic portion of F1 histone, consequent disruptions of lysine-phosphate interactions, and increased susceptibility to polyelectrolyte expansion.

Interaction of lysine-rich histones and DNA

Abstract Complexes of lysine-rich histones (f1) with native linear calf thymus DNA were prepared in soluble form by a salt-gradient dialysis method and examined by a variety of physico-chemical methods. Absorption spectroscopy and circular dichroism revealed minimal differences between DNA and histone-DNA complexes, suggesting that the DNA remains in the B conformation with histone present. Thermal denaturation and thermal renaturation studies indicated that the presence of histone resulted in a marked stabilization of DNA structure and permitted rapid renaturation of denatured regions. Maximum renaturation was effected with as few as 20 to 40 histone molecules per DNA molecule (107 mol. wt). Sedimentation velocity measurements of the complexes indicated that, at infinite dilution, the particles behave as single DNA molecules with associated histones. Evidence for concentration-dependent aggregation of the histone-DNA particles was also obtained. Dye-binding studies suggested that lysine-rich histone does not bind in the small groove of DNA; the presence of histone on DNA does not inhibit actinomycin binding. Furthermore, evidence was obtained indicating that histone might bind within the large groove of DNA; histone complexes with unglucosylated T2∗ DNA were less effective substrates for glucosylation by α-glucosyl transferase than was T2∗ DNA.

The binding of nuclear non-histone protein to DNA

Abstract The binding of rat liver nuclear non-histone proteins to DNA has been detected using a membrane filter technique. Binding is optimal at 0.05 M NaCl, increases with decreasing pH, and is enhanced in the presence of Mg2+. About 50% of the DNA is bound reversibly. Histone contamination of non-histone preparations contributes little, if at all, to the observed binding while cytoplasmic proteins, isolated like non-histones, have no ability to bind DNA. The binding activity, which is unstable, can be destroyed by heat or pronase.

Replication bands and nucleoli in the macronucleus of Euplotes eurystomus: an ultrastructural and cytochemical study

The principal structural compartments of the macronucleus of Euplotes eurystomus were examined by ultrastructural and cytochemical procedures. Interphase chromatin is condensed in highly compact granules that stain intensely with the DNA‐specific osmium‐amine procedure. Nucleoli react strongly with silver and with thiol‐specific reagents, but are almost completely unstained by osmium‐amine. The organelle of DNA synthesis, the replication band, is composed of 2 zones. The forward zone consists of highly ordered chromatin fibers, stains strongly with osmium‐amine, with silver, and with thiol‐specific reagents. The rear zone, which is the site of DNA synthesis, is impoverished in DNA, and is very sensitive to collapse induced by in vivo heat shock, or during nuclear isolation.

Differential distribution of α-tubulin isotypes in Euplotes eurystomus determined by confocal immunofluorescence microscopy**

Detergent permeabilized Euplotes eurystomus (a fresh water hypotrichous ciliate) was reacted with monoclonal and polyclonal antibodies specific for either detyrosinated or tyrosinated α‐tubulin (Glu‐ or Tyr‐tubulin). The isolated cytoskeleton‐nuclear complex was examined by Western immunoblotting and by immunofluorescent and electron microscopic methods. Both Glu‐ and Tyr‐tubulins were detected by immunoblot analysis. Immunofluorescent microscopy indicated that the α‐tubulin isotypes are concentrated in different regions of permeabilized cells: Glu‐tubulin is located primarily in cirri, membranelles, and surrounding the macro‐ and micronuclei. Tyr‐tubulin is principally at the bases of cirri and membranelles. This differential distribution of α‐tubulin isotypes is discussed in terms of current concepts concerning the correlation of tubulin post‐translational modifications to microtubule stability. Confocal immunofluorescent imaging was of critical importance in clearly differentiating the Glu‐tubulin isotype surrounding the macro‐ and micronuclei from a brilliantly fluorescent environment originating from cytoskeletal structures. In conjunction with conventional and stereo‐electron microscopy, confocal optical microscopy provided convincing evidence for a “basket” of microtubules surrounding both nuclei.

Electron microscope tomography of Balbiani Ring hnRNP substructure

Three-dimensional (3-D) reconstructions, by electron microscope tomography, of selectively stained, contrast enhanced Balbiani Ring (BR) hnRNP granules reveal a complex spatial arrangement of RNA-rich domains. This particulate substructure was examined by volume rendering computer graphics. Modeling the arrangement of RNA-rich domains is made difficult by apparent structural flexibility and/or heterogeneity of composition. Formulation of a consensus 3-D arrangement of RNA-rich domains will require an expanded data base of reconstructed BR granules and the development of new image manipulation and analysis techniques. This study demonstrates the potential for ultra-structural cell biology of combining several new techniques: selective nucleic acid staining, electron spectroscopic imaging to enhance contrast, electron microscope tomography and volume rendering computer graphics.

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.

Cytochemistry of the chromatin replication band in hypotrichous ciliated protozoa staining with silver and thiol-specific coumarin maleimide

A modification of the silver-staining techniques for nucleolar organizing regions (NORs) was used to stain selectively the macronuclear replication bands (RBs) and nucleoli in hypotrichous ciliated protozoa (Euplotes, Stylonychia, and Oxytricha). Silver staining of both types of structures was trypsin-sensitive and DNase I-insensitive, suggesting the involvement of proteins. Silver-staining proteins in the RB were differentially extracted with acid, without any decrease in nucleolar staining. Triton-acid-urea gel electrophoresis of an acid extract of Euplotes macronuclei revealed enhanced silver reaction with a single protein upon selective silver staining. An abundance of thiol groups was also demonstrated in the RBs and nucleoli by the fluorochrome 3-(4-maleimidylphenyl)-7-diethylamino-4-methyl coumarin (coumarin maleimide). Histochemical studies, including blocking thiols with N-ethyl maleimide (NEM), indicated that thiols were not necessary for silver staining, and that proteins in the RBs and nucleoli reacting with coumarin maleimide were not acid extractable.

Lectin‐like components in the macronuclear replication bands of Euplotes eurystomus

Upon incubation with fluoresceinylated neoglycoproteins, isolated macronuclei from the ciliated protozoan Euplotes eurystomus display different labelling patterns depending on the nature of the sugar bound to the neoglycoproteins. Specific sugar‐binding components (i.e., lectin‐like molecules) are associated with presumed nucleoli and with the macronuclear replication bands. This is the first demonstration that DNA synthesis and sugar‐binding components are co‐localized in an eukaryotic cell.