Ada L. 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.

Synthesis of a more stable osmium ammine electron-dense DNA stain.

Specific DNA staining for electron microscopic observation is simplified by a shorter synthesis of the staining reagent. The new, more reliable reagent, osmium ammine-B, is stable for more than a year, dissolves completely in water, and does not require reoptimization of staining conditions for every batch, yet reproducibly gives strong contrast to DNA-containing structures.

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.

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.

Localization of acetylated histone H4 in the macronucleus of Euplotes

Antisera specific to acetylated and unacetylated N-terminal domains of histone H4 were employed to map the spatial distribution of exposed antigenic determinants in the macronucleus of Euplotes eurystomus. Sites of putative transcription-related acetylation appear to reside in the regions between condensed chromatin granules and disappear during chromatin restructuring in the forward zone of replication bands. Coincident with synthesis of newly replicated chromatin, the specific antisera reveal a resumption of exposed sites of H4 acetylation in the rear zone of replication bands.

Inhibition of Dna Synthesis In the Macronuclear Replication Band of Euplotes Eurystomus

ABSTRACT. The replication band is a large, migrating, macronuclear domain that is the site of DNA synthesis in hypotrichous ciliated protozoa. A number of agents that produce inactivation of this structure and its replicational activity are described here. These agents include heat shock, aphidicolin, cell crowding, various cAMP phosphodiesterase inhibitors and a calmodulin inhibitor. With the exception of aphidicolin, which has a direct inhibitory effect upon DNA polymerases, the mechanisms of inactivation are presently unknown. the inactivating properties of cAMP phosphodiesterase inhibitors suggest that intracellular cAMP levels may influence replication band structure and function.

Chapter 5 Visualization of Chromatin v-Bodies

Publisher Summary This chapter discusses the methods for visualization of chromatin v bodies. Many stains can be used successfully to visualize v bodies. Ethanol and methanol solvents do not affect the general morphology of the v bodies at this stage. Positive stains give excellent contrast but do not resolve v bodies that are in contact with each other. High-concentration negative stains (1% or more) have too much contrast to visualize the small amounts of stain among close particles. Low-concentration negative stains produce less contrast but tend to be more delicate and show more detail. In this study, the standard and most reliable staining procedure at present is to put a small drop of 0.01 M uranyl acetate in water, on the sample side of the grid for 30 seconds and touch the edge of the grid to the edge of bibulous paper to drain as thoroughly as possible. Low-angle rotary shadowing has been used successfully in several laboratories. It is a high-contrast method and makes the electron microscopy much more rapid. However, the details of the internal v -body structure are lost, and subtle differences in morphology are unattainable.