DNA is replicated at the nuclear cage

1980 ◽  
Vol 46 (1) ◽  
pp. 365-386 ◽  
Author(s):  
S.J. McCready ◽  
J. Godwin ◽  
D.W. Mason ◽  
I.A. Brazell ◽  
P.R. Cook
Keyword(s):  
Electron Microscopy ◽  
Dna Synthesis ◽  
Hela Cells ◽  
Membrane Structure ◽  
Structural Unit ◽  
Nuclear Dna ◽  
Short Pulses ◽  
Restriction Endonuclease Ecori ◽  
Endonuclease Ecori ◽  
Average Size

Structures resembling nuclei are released when HeLa cells are lysed in a detergent and 2 M salt. These nucleoids, which lack any organized membrane structure, contain all the nuclear DNA packaged within a cage of RNA and protein. Their DNA is supercoiled so that the linear DNA must remain unbroken and looped during lysis. Following digestion with the restriction endonuclease, EcoRI, cages and associated DNA were filtered free of unattached DNA. Pulse-labelled (i.e. newly synthesized) DNA remains preferentially associated with the cages. This association has been confirmed by autoradiography. When nucleoids are prepared for electron microscopy by the Kleinschmidt procedure the DNA spills out to form a skirt around the flattened cage. Labelling, which is restricted to the region of the cage after short pulses, extends out into the skirt as the labelling time increases. A model, based on the premise that replication takes place at the nuclear cage, is presented in the Appendix. The results of the biochemical experiments and electron microscopy both indicate that the average size of the unit of replication is approximately 2 micrometer. This is about one-quarter the size of the average structural unit - the loop. Therefore sequences in the loop must become attached to the nuclear cage prior to the initiation of DNA synthesis.

scholarly journals Analysis of DNA attached to the chromosome scaffold.

1982 ◽  
Vol 93 (2) ◽  
pp. 278-284 ◽  
Author(s):  
M T Kuo
Keyword(s):  
Dna Sequences ◽  
Southern Blotting ◽  
Nuclear Dna ◽  
Micrococcal Nuclease ◽  
Nonhistone Protein ◽  
Sequence Class ◽  
Restriction Endonuclease Ecori ◽  
Cold Spring ◽  
Endonuclease Ecori ◽  
Chromosome Scaffold

Two different methods have been described to investigate whether any specific DNA sequences are intimately associated with the metaphase chromosome scaffold. The chromosome scaffold, prepared by dehistonization of chromosomes with 2 M NaCl, is a nonhistone protein complex to which many looped DNA molecules are attached (Laemmli et al., 1977, Cold Spring Harbor Symp. Quant. Biol. 42:351--360). Chromosome scaffold DNA was prepared from dehistonized chicken MSB chromosomes by restriction endonuclease EcoRI digestion followed by removal of the looped DNA by sucrose gradient sedimentation. Alternatively, the scaffold DNA was prepared from micrococcal nuclease-digested intact chromosomes using sucrose gradients containing 2M NaCl. Solution hybridization of the radioactively labeled scaffold DNA with a large excess of total nuclear DNA revealed that, in either case, the scaffold DNA is not a unique sequence class of genomic DNA. Southern-blotting hybridization also showed that the scaffold DNA prepared from EcoRI-digested dehistonized chromosomes was not enriched (or depleted) in the ovalbumin gene sequences. The possibility of a dynamic interaction of protein and DNA in the chromosome scaffold and the possibility that the scaffold is a preparative artifact are discussed.

scholarly journals Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

1981 ◽  
Vol 199 (2) ◽  
pp. 453-455 ◽  
Author(s):  
N Hardt ◽  
G Pedrali-Noy ◽  
F Focher ◽  
S Spadari
Keyword(s):  
Dna Repair ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Hela Cells ◽  
Nuclear Dna ◽  
Detectable Effect ◽  
Repair Synthesis ◽  
Dna Polymerase Alpha ◽  
Dna Repair Synthesis

A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.

scholarly journals Microbubbles in replicating nuclear deoxyribonucleic acid from Physarum polycephalum

1979 ◽  
Vol 183 (2) ◽  
pp. 477-480 ◽  
Author(s):  
D A F Gillespie ◽  
N Hardman
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Dna Synthesis ◽  
Physarum Polycephalum ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
S Phase ◽  
Dna Structures ◽  
Nuclear Deoxyribonucleic Acid

Clusters of microbubbles, represent probable sites of newly initiated DNA synthesis, were identified in nuclear DNA from Physarum polycephalum by using the electron microscope. Their presence is associated specifically with S-phase. Each microbubble corresponds in size to a replicating segment of DNA about 100-5000 nucleotide residues in length. The DNA structures containing microbubbles are metastable, and revert to native DNA in the presence of moderate concentrations of formamide used to prepare samples for electron microscopy. It is suggested that each cluster of microbubbles may correspond to a unit of replication (a replicon) in Physarum DNA.

scholarly journals The X-ray, Raman and TEM Signatures of Cellulose-Derived Carbons Explained

2022 ◽  
Vol 8 (1) ◽  
pp. 4
Author(s):  
Petros Kasaira Mubari ◽  
Théotime Beguerie ◽  
Marc Monthioux ◽  
Elsa Weiss-Hortala ◽  
Ange Nzihou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Raman Spectroscopy ◽  
Structural Unit ◽  
X Ray Diffraction ◽  
X Ray ◽  
Characterization Techniques ◽  
Transmission Electron ◽  
Crystallite Sizes ◽  
Xrd Patterns ◽  
Average Size

Structural properties of carbonized cellulose were explored to conjugate the outcomes from various characterization techniques, namely X-ray diffraction (XRD), Raman spectroscopy, and high-resolution transmission electron microscopy. All these techniques have evidenced the formation of graphene stacks with a size distribution. Cellulose carbonized at 1000 and 1800 °C at a heating rate of 2 °C/min showed meaningful differences in Raman spectroscopy, whereas in XRD, the differences were not well pronounced, which implies that the crystallite sizes calculated by each technique have different significations. In the XRD patterns, the origin of a specific feature at a low scattering angle commonly reported in the literature but poorly explained so far, was identified. The different approaches used in this study were congruous in explaining the observations that were made on the cellulose-derived carbon samples. The remnants of the basic structural unit (BSU) are developed during primary carbonization. Small graphene-based crystallites inherited from the BSUs, which formerly developed during primary carbonization, were found to coexist with larger ones. Even if the three techniques give information on the average size of graphenic domains, they do not see the same characteristics of the domains; hence, they are not identical, nor contradictory but complementary. The arguments developed in the work to explain which characteristics are deduced from the signal obtained by each of the three characterization techniques relate to physics phenomena; hence, they are quite general and, therefore, are valid for all kind of graphenic materials.

The effect of hydroxyurea on DNA synthesis of Tipula iridescent virus and Estigmene acrea cells

1983 ◽  
Vol 29 (2) ◽  
pp. 254-260 ◽  
Author(s):  
Malgorzata Kloc ◽  
Peter E. Lee
Keyword(s):  
Electron Microscopy ◽  
Dna Synthesis ◽  
Light Microscope ◽  
Nuclear Dna ◽  
Stages Of Development ◽  
Iridescent Virus ◽  
Estigmene Acrea ◽  
Microscope Autoradiography

Viral-specific DNA synthesis of Tipula iridescent virus (TIV) was not affected in Estigmene acrea cells which were continuously exposed to 300 μg/mL hydroxyurea (HU) as detected by light microscope autoradiography. Electron microscopy of such cells showed viroplasmic centres with virions in various stages of development. In nor mal cells similarly exposed to HU, nuclear DNA synthesis was reduced by 70–80%.

scholarly journals Differential Effect of Ethidium Bromide and Cytosine Arabinoside on Mitochondrial and Nuclear DNA Synthesis in HeLa Cells

1972 ◽  
Vol 27 (8) ◽  
pp. 989-991 ◽  
Author(s):  
Kenzo Kato ◽  
Klaus D. Radsak ◽  
Hilary Koprowski
Keyword(s):  
Hela Cell ◽  
Dna Synthesis ◽  
Ethidium Bromide ◽  
Hela Cells ◽  
Cytosine Arabinoside ◽  
Differential Effect ◽  
Mammalian Cells ◽  
Nuclear Dna ◽  
Circular Dna ◽  
Isopycnic Centrifugation

The effect of ethidium bromide (EB) on the synthesis of circular DNA of mammalian cells was studied by isopycnic centrifugation in a CsCl-EB solution. EB (0.1—0.5 μg/ml) interferes with the synthesis of newly-formed circular DNA of HeLa cell mitochondria and causes degradation of the pre-existing circular DNA, as well. Under the same conditions, nuclear DNA synthesis was not inhibited. This effect was not reversible at a concentration of 0.5 μg EB/ml or more. Cytosine arabinoside (ara-C) did not exhibit an effect similar to that of EB.

scholarly journals Lesions induced in DNA by ultraviolet light are repaired at the nuclear cage

1984 ◽  
Vol 70 (1) ◽  
pp. 189-196
Author(s):  
S.J. McCready ◽  
P.R. Cook
Keyword(s):  
Dna Synthesis ◽  
Ultraviolet Light ◽  
Hela Cells ◽  
Nuclear Matrix ◽  
Ultraviolet Irradiation ◽  
Mammalian Cells ◽  
Nuclear Dna ◽  
S Phase ◽  
Unscheduled Dna Synthesis ◽  
Phase Synthesis

In mammalian cells, S-phase DNA synthesis occurs at sites fixed to a sub-nuclear structure, the nuclear matrix or cage. This is an ordered network of non-histone proteins, which maintains its essential morphology even in the absence of DNA. We show here that unscheduled DNA synthesis following exposure of HeLa cells to ultraviolet light also takes place at this sub-structure. We also show that ultraviolet irradiation grossly reorganizes nuclear DNA, arresting S-phase synthesis at the cage and leaving the residual synthesis highly localized.

Platinum Compounds as Stains for Electron Microscopy

1974 ◽  
Vol 32 ◽  
pp. 230-231
Author(s):  
S. K. Aggarwal ◽  
P. McAllister ◽  
R. W. Wagner ◽  
B. Rosenberg
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Uranyl Acetate ◽  
Sarcoma 180 ◽  
Platinum Compounds ◽  
Kb Cells ◽  
En Bloc ◽  
Human Lymphoma ◽  
Ultrathin Sections ◽  
Blue Coloration

Uranyl acetate has been used as an electron stain for en bloc staining as well as for staining ultrathin sections in conjunction with various lead stains (Fig. 1). Present studies reveal that various platinum compounds also show promise as electron stains. Certain platinum compounds have been shown to be effective anti-tumor agents. Of particular interest are the compounds with either uracil or thymine as one of the ligands (cis-Pt(II)-uracil; cis-Pt(II)-thymine). These compounds are amorphous, highly soluble in water and often exhibit an intense blue coloration. These compounds show enough electron density to be used as stains for electron microscopy. Most of the studies are based on various cell lines (human AV, cells, human lymphoma cells, KB cells, Sarcoma-180 ascites cells, chick fibroblasts and HeLa cells) while studies on tissue blocks are in progress.

Electron Microscopy of Mycoplasma Pneumoniae

1971 ◽  
Vol 29 ◽  
pp. 258-259 ◽  
Author(s):  
E. S. Boatman ◽  
G. E. Kenny
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Growth Phase ◽  
Hela Cells ◽  
Sulfonic Acid ◽  
Gamma Globulin ◽  
Horse Serum ◽  
Morphological Aspects ◽  
The Family

Information concerning the morphology and replication of organism of the family Mycoplasmataceae remains, despite over 70 years of study, highly controversial. Due to their small size observations by light microscopy have not been rewarding. Furthermore, not only are these organisms extremely pleomorphic but their morphology also changes according to growth phase. This study deals with the morphological aspects of M. pneumoniae strain 3546 in relation to growth, interaction with HeLa cells and possible mechanisms of replication.The organisms were grown aerobically at 37°C in a soy peptone yeast dialysate medium supplemented with 12% gamma-globulin free horse serum. The medium was buffered at pH 7.3 with TES [N-tris (hyroxymethyl) methyl-2-aminoethane sulfonic acid] at 10mM concentration. The inoculum, an actively growing culture, was filtered through a 0.5 μm polycarbonate “nuclepore” filter to prevent transfer of all but the smallest aggregates. Growth was assessed at specific periods by colony counts and 800 ml samples of organisms were fixed in situ with 2.5% glutaraldehyde for 3 hrs. at 4°C. Washed cells for sectioning were post-fixed in 0.8% OSO4 in veronal-acetate buffer pH 6.1 for 1 hr. at 21°C. HeLa cells were infected with a filtered inoculum of M. pneumoniae and incubated for 9 days in Leighton tubes with coverslips. The cells were then removed and processed for electron microscopy.

Grain Refinement in Titanium-Erbium Alloys

1974 ◽  
Vol 32 ◽  
pp. 522-523
Author(s):  
B. B. Rath ◽  
J. E. O'Neal ◽  
R. J. Lederich
Keyword(s):  
Electron Microscopy ◽  
Size Distribution ◽  
Grain Refinement ◽  
Grain Growth ◽  
Volume Fraction ◽  
Pure Titanium ◽  
Systematic Investigation ◽  
Second Phase ◽  
Titanium Matrix ◽  
Average Size

Addition of small amounts of erbium has a profound effect on recrystallization and grain growth in titanium. Erbium, because of its negligible solubility in titanium, precipitates in the titanium matrix as a finely dispersed second phase. The presence of this phase, depending on its average size, distribution, and volume fraction in titanium, strongly inhibits the migration of grain boundaries during recrystallization and grain growth, and thus produces ultimate grains of sub-micrometer dimensions. A systematic investigation has been conducted to study the isothermal grain growth in electrolytically pure titanium and titanium-erbium alloys (Er concentration ranging from 0-0.3 at.%) over the temperature range of 450 to 850°C by electron microscopy.

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