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A L Olins

Publications and source records attributed to A L Olins.

At least 37 records · Page 2Linked to original sources
PubMed1987

In vitro DNA synthesis in the macronuclear replication band of Euplotes eurystomus.

Isolated macronuclei from the hypotrichous ciliated protozoan Euplotes eurystomus incorporate biotinylated dUTP specifically into the replication band (RB) as detected with immunofluorescence, using rabbit anti-biotin antibodies followed by fluorescein-conjugated goat anti-rabbit IgG. When gold-conjugated goat anti-rabbit IgG was used in a preembedded reaction, subsequent immunoelectron microscopic analysis demonstrated that the biotinylated nucleotide appeared more concentrated in the rear zone of the RB, with almost no labeling in the forward zone. It was possible to use the immunofluorescent assay to establish that incorporation of biotinylated dUTP is inhibited by simultaneous addition of N-ethyl maleimide or aphidicolin, and by omission of any one of the other unlabeled dNTPs. In addition, prolonged heat shock of the intact cells, before lysis and in vitro assay, yielded markedly reduced incorporation. Comparison with published data on the in vivo incorporation of [3H]thymidine into Euplotes eurystomus RBs indicates the fidelity of the in vitro reaction.

Animals↗
PubMed1987

The varifocal mirror for 3-D display of electron microscope tomography.

Three-dimensional reconstruction by tomographic procedures of objects viewed with transmission electron microscopy generates a volume of densities arrayed in raster format. This volume can be conveniently displayed and analysed employing a varifocal mirror (VFM) and an associated image processing system. We have developed a number of interactive programs useful to display and analyse the 3-D data, including depth windowing, intensity mapping, image zoom, image rotation and cursor overlay. Examples of these functions are presented, employing data from electron microscope tomography (EMT) of a transcriptionally-active eukaryotic gene, the Balbiani ring of Chironomus polytene chromosomes.

Animals↗
PubMed1986

Subfractionation of soluble macronuclear chromatin and enrichment of specific genes as chromatin from Euplotes eurystomus.

Euplotes eurystomus is a hypotrichous ciliated protozoan possessing within one cytoplasm a transcriptionally-inactive micronucleus with chromosomal-size DNA and a transcriptionally active macronucleus with "gene-size" DNA fragments. The chromatin in the macronucleus can be isolated in a soluble form without prior treatment by nucleases. In this study, macronuclear, soluble chromatin was subfractionated using isokinetic sucrose density gradient ultracentrifugation in a buffer consisting of 50 mM NaCl, 1 mM Na2 EDTA, 1 mM TEA HCl, pH 7.0, 0.1 mM TLCK and 0.1 mM PMSF. Fractions were collected and analyzed by DNA and protein gel electrophoresis, dot blot hybridization with specific gene probes, and modified Miller chromatin spreads. Analysis of the chromatin spreads showed that the sizes of the chromatin fragments in the various fractions correlate with the DNA size of the fragments. When dot blots of the fractions were hybridized with 5S rRNA, tubulin and rRNA gene probes we obtained about a 6 to 14-fold enrichment of hybridizable sequences in individual fractions. There appear to be differences in the non-histones present on each fraction as well as some overall similarities in histone and non-histone proteins.

Animals↗
PubMed1986

Preparation and characterization of soluble macronuclear chromatin from the hypotrich Euplotes eurystomus.

Euplotes eurystomus is a hypotrichous ciliate containing a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus. Soluble MAC chromatin contains a normal complement of inner histones, an H1-like protein which is very sensitive to proteolysis, and a considerable proportion of non-histone proteins. A combination of N-Tosyl lysine chloromethyl ketone (TLCK) and PMSF was found to be most effective in preventing proteolysis. Microccocal nuclease digestion yielded an average nucleosome repeat length of 187 +/- 25 bp for soluble chromatin; and 190 +/- 9 bp for isolated macronuclei. Thermal denaturation profiles of MAC chromatin in 0.25 mM EDTA display two main transitions at about 76 and 83 degrees C, resembling the melting of soluble chicken erythrocyte chromatin. Circular dichroic spectra of MAC chromatin were compared to soluble chicken erythrocyte chromatin under the same ionic strength conditions and were found to be very similar. 2D chromatin/DNA agarose gel electrophoresis resulted in a diagonal line of DNA staining, which establishes a strict correlation between DNA and chromatin electrophoretic mobility.

Animals↗
PubMed1986

Examination of the macronuclear replication band in Euplotes eurystomus with monoclonal antibodies.

A panel of eight monoclonal antibodies (MAbs) was prepared from spleen cells of mice immunized with macronuclear replication bands (RBs) isolated from Euplotes eurystomus. Antibodies were investigated with a solid phase radioimmunoassay (RIA) using either soluble chromatin from isolated RBs or from total macronuclei as antigen. The RIA showed that several MAbs recognized antigens present only in the RB or macronucleus, whereas others recognized antigens present in both structures. Specificity of the MAbs was also examined by indirect immunofluorescence. Antibody C10 recognized an antigen in the rear zone of the RB, whereas MAbs G6 and B2 appeared to stain both the forward and rear zones of the RB. Antibody A7 recognized an epitope distributed throughout the macronucleus except in the RB. Cytochemical studies with degradative enzymes suggested that antigens localized by immunofluorescence were composed of proteins. Immunoblots of SDS PAGE permitted identification of a few proteins that reacted with three of the RB-specific MAbs. Monoclonal antibodies that identify the presence or absence of reactivity of specific proteins in the RB could prove useful in the study of chromatin structure and the mechanism of chromatin replication.

Animals↗
PubMed1986

DNA compaction during intense transcription measured by electron microscope tomography.

Thick sections (0.5-1.0 micron) containing pilocarpine-hyperstimulated Balbiani ring (BR) genes of Chironomus tentans salivary glands were reconstructed by electron microscope tomography (EMT). Six transcription loops were analyzed at low resolution permitting a measurement of transcription unit length in situ. Comparison of the average transcription unit length with the BR gene coding region DNA length yields an estimate of the DNA compaction ratio in situ. The estimated DNA compaction ratio (approximately 3-fold) is consistent with a significant degree of nucleosome unfolding in the hyperstimulated BR genes.

Chironomidae↗
PubMed1985

Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus.

A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.

Animals↗
PubMed1984

Modeling Balbiani Ring gene transcription with electron microscope tomography.

Cross-sectional views of mature regions of Balbiani Ring (BR) transcription loops were reconstructed by electron microscope tomography. Balsa wood models were built based upon the resulting tomograms. Coordinates of the centers of the ribonucleoprotein BR granules were estimated and employed to calculate the approximate orientation of the BR transcription unit axis in the plastic section. The density of BR granules per micron of transcription unit axis was estimated. Employing structural parameters for mature regions of BR transcription loops derived from present and previous studies, theoretical calculations were performed to examine potential steric restrictions around the central chromatin axis. Tomograms were also observed and photographed on a varifocal (vibrating) mirror to illustrate the utility of a rapid user-interactive 3-D display.

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

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

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

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↗