scholarly journals ACCUMULATION OF REPLICATIVE INTERMEDIATES OF MITOCHONDRIAL DNA IN TETRAHYMENA PYRIFORMIS GROWN IN ETHIDIUM BROMIDE

1974 ◽  
Vol 61 (2) ◽  
pp. 383-397 ◽  
Author(s):  
William B. Upholt ◽  
Piet Borst
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Tetrahymena Pyriformis ◽  
Linear Molecule ◽  
Chain Growth ◽  
Predominant Species ◽  
Velocity Gradients ◽  
Large Numbers ◽  
Replicative Intermediates

The effect of growth of Tetrahymena pyriformis in ethidium bromide (EthBr) on the structure and synthesis of mitochondrial DNA (mtDNA) has been investigated. During the first 5 h of growth in EthBr, mtDNA synthesis is inhibited 95% or more. After 10–15 h, this block is partially released and large numbers of replicating molecules accumulate, indicating that inhibition by EthBr primarily affects the rate of chain growth and not the initiation of new rounds of replication. The accumulated molecules sediment more rapidly than normal Tetrahymena mtDNA and do not contain enough single-strand regions to distinguish them from normal Tetrahymena mtDNA when banded in buoyant CsCl or NaI gradients. Electron microscopy shows that the predominant species in this rapidly sedimenting DNA is a linear molecule containing one symmetrical double-stranded replication loop of varying size located at its center. No degradation of mtDNA from cells grown in EthBr was detected in alkaline velocity gradients.

Mitochondrial DNA from 4-Cell Stages of Ascaris Lumbricoides

1974 ◽  
Vol 16 (3) ◽  
pp. 593-601
Author(s):  
H. TOBLER ◽  
C. GUT
Keyword(s):  
Electron Microscopy ◽  
Mitochondrial Dna ◽  
Ascaris Lumbricoides ◽  
Buoyant Density ◽  
Contour Length ◽  
Cell Stage ◽  
Replicative Intermediates ◽  
Dna Amounts

Mitochondrial DNA (mtDNA) has been isolated from 4-cell stages of Ascaris lumbricoides. This DNA amounts to about 40% of the total quantity of 4-cell-stage DNA. Its buoyant density in neutral CsCl gradients is 1.686 g cm-3. Electron microscopy of mtDNA demonstrated the presence of circular molecules with an average contour length of 4.64 µm. About 15% of these molecules are supercoiled, covalently closed circles, whereas some 2% consist of double-forked circular molecules. The form and size of these branched molecules suggest that they are replicative intermediates.

Evaluation of the dark field method for large association of spread DNA

1978 ◽  
Vol 36 (2) ◽  
pp. 190-191
Author(s):  
Douglas C. Barker
Keyword(s):  
Electron Microscopy ◽  
Molecular Weight ◽  
Mitochondrial Dna ◽  
Extraction Method ◽  
Field Method ◽  
Dark Field ◽  
Kinetoplast Dna ◽  
Field Electron ◽  
Field Electron Microscopy ◽  
Dark Field Electron

A number of satisfactory methods are available for the electron microscopy of nicleic acids. These methods concentrated on fragments of nuclear, viral and mitochondrial DNA less than 50 megadaltons, on denaturation and heteroduplex mapping (Davies et al 1971) or on the interaction between proteins and DNA (Brack and Delain 1975). Less attention has been paid to the experimental criteria necessary for spreading and visualisation by dark field electron microscopy of large intact issociations of DNA. This communication will report on those criteria in relation to the ultrastructure of the (approx. 1 x 10-14g) DNA component of the kinetoplast from Trypanosomes. An extraction method has been developed to eliminate native endonucleases and nuclear contamination and to isolate the kinetoplast DNA (KDNA) as a compact network of high molecular weight. In collaboration with Dr. Ch. Brack (Basel [nstitute of Immunology), we studied the conditions necessary to prepare this KDNA Tor dark field electron microscopy using the microdrop spreading technique.

Properties of Isolated Mitochondria of Agaricus Bisporus: Their Relationship to Dormancy and the Germination of Spores

1973 ◽  
Vol 31 ◽  
pp. 458-459
Author(s):  
K. S. McCarty ◽  
R. F. Weave ◽  
L. Kemper ◽  
F. S. Vogel
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Microscopic Examination ◽  
Agaricus Bisporus ◽  
Osmotic Shock ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Isolated Mitochondria ◽  
Dna Strands ◽  
Cytoplasmic Ribosomes

During the prodromal stages of sporulation in the Basidiomycete, Agaricus bisporus, mitochondria accumulate in the basidial cells, zygotes, in the gill tissues prior to entry of these mitochondria, together with two haploid nuclei and cytoplasmic ribosomes, into the exospores. The mitochondria contain prominent loci of DNA [Fig. 1]. A modified Kleinschmidt spread technique1 has been used to evaluate the DNA strands from purified whole mitochondria released by osmotic shock, mitochondrial DNA purified on CsCl gradients [density = 1.698 gms/cc], and DNA purified on ethidium bromide CsCl gradients. The DNA appeared as linear strands up to 25 u in length and circular forms 2.2-5.2 u in circumference. In specimens prepared by osmotic shock, many strands of DNA are apparently attached to membrane fragments [Fig. 2]. When mitochondria were ruptured in hypotonic sucrose and then fixed in glutaraldehyde, the ribosomes were released for electron microscopic examination.

Selective Sampling and Orientation of Non-Homogeneous Tissues

1968 ◽  
Vol 26 ◽  
pp. 98-99
Author(s):  
C. C. Clawson ◽  
L. W. Anderson ◽  
R. A. Good
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscopy ◽  
Random Sampling ◽  
New Method ◽  
Microscope Examination ◽  
Small Areas ◽  
Selective Sampling ◽  
Large Numbers ◽  
Specific Orientation

Investigations which require electron microscope examination of a few specific areas of non-homogeneous tissues make random sampling of small blocks an inefficient and unrewarding procedure. Therefore, several investigators have devised methods which allow obtaining sample blocks for electron microscopy from region of tissue previously identified by light microscopy of present here techniques which make possible: 1) sampling tissue for electron microscopy from selected areas previously identified by light microscopy of relatively large pieces of tissue; 2) dehydration and embedding large numbers of individually identified blocks while keeping each one separate; 3) a new method of maintaining specific orientation of blocks during embedding; 4) special light microscopic staining or fluorescent procedures and electron microscopy on immediately adjacent small areas of tissue.

Albumin-Induced Endocytic Vacuoles in Tetrahymena Pyrijormis

1975 ◽  
Vol 33 ◽  
pp. 694-695
Author(s):  
L. J. Brenner ◽  
D. G. Osborne ◽  
B. L. Schumaker
Keyword(s):  
Electron Microscopy ◽  
Bovine Serum Albumin ◽  
Bovine Serum ◽  
Protein Concentration ◽  
Tetrahymena Pyriformis ◽  
Sequence Of Events ◽  
Cytoplasmic Bodies ◽  
Food Vacuoles ◽  
Mouse Ascites ◽  
Normal Human

Exposure of the ciliate, Tetrahymena pyriformis, strain WH6, to normal human or rabbit sera or mouse ascites fluids induces the formation of large cytoplasmic bodies. By electron microscopy these (LB) are observed to be membrane-bounded structures, generally spherical and varying in size (Fig. 1), which do not resemble the food vacuoles of cells grown in proteinaceous broth. The possibility exists that the large bodies represent endocytic vacuoles containing material concentrated from the highly nutritive proteins and lipoproteins of the sera or ascites fluids. Tetrahymena mixed with bovine serum albumin or ovalbumin solutions having about the same protein concentration (7g/100 ml) as serum form endocytic vacuoles which bear little resemblance to the serum-induced LB. The albumin-induced structures (Fig. 2) are irregular in shape, rarely spherical, and have contents which vary in density and consistency. In this paper an attempt is made to formulate the sequence of events which might occur in the formation of the albumin-induced vacuoles.

Development of a mito-CRISPR system for generating mitochondrial DNA-deleted strain in Saccharomyces cerevisiae

2020 ◽  
Vol 85 (4) ◽  
pp. 895-901
Author(s):  
Takamitsu Amai ◽  
Tomoka Tsuji ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Nuclear Genome ◽  
Target Sequence ◽  
Guide Rna ◽  
Crispr System ◽  
Mitochondrial Target ◽  
Gene Treatment ◽  
Mtdna Deletion

ABSTRACT Mitochondrial dysfunction can occur in a variety of ways, most often due to the deletion or mutation of mitochondrial DNA (mtDNA). The easy generation of yeasts with mtDNA deletion is attractive for analyzing the functions of the mtDNA gene. Treatment of yeasts with ethidium bromide is a well-known method for generating ρ° cells with complete deletion of mtDNA from Saccharomyces cerevisiae. However, the mutagenic effects of ethidium bromide on the nuclear genome cannot be excluded. In this study, we developed a “mito-CRISPR system” that specifically generates ρ° cells of yeasts. This system enabled the specific cleavage of mtDNA by introducing Cas9 fused with the mitochondrial target sequence at the N-terminus and guide RNA into mitochondria, resulting in the specific generation of ρ° cells in yeasts. The mito-CRISPR system provides a concise technology for deleting mtDNA in yeasts.

scholarly journals Velocity Gradient Separation Reveals a New Extracellular Vesicle Population Enriched in miR-155 and Mitochondrial DNA

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 526
Author(s):  
Myriam Vaillancourt ◽  
Audrey Hubert ◽  
Caroline Subra ◽  
Julien Boucher ◽  
Wilfried Wenceslas Bazié ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Velocity Gradient ◽  
Messenger Rna ◽  
Sucrose Density Gradient ◽  
Extracellular Vesicle ◽  
Therapeutic Interventions ◽  
Optimal Separation ◽  
Velocity Gradients ◽  
Commercial Kits ◽  
Hiv 1

Extracellular vesicles (EVs) and their contents (proteins, lipids, messenger RNA, microRNA, and DNA) are viewed as intercellular signals, cell-transforming agents, and shelters for viruses that allow both diagnostic and therapeutic interventions. EVs circulating in the blood of individuals infected with human immunodeficiency virus (HIV-1) may provide insights into pathogenesis, inflammation, and disease progression. However, distinguishing plasma membrane EVs from exosomes, exomeres, apoptotic bodies, virions, and contaminating proteins remains challenging. We aimed at comparing sucrose and iodixanol density and velocity gradients along with commercial kits as a means of separating EVs from HIV particles and contaminating protein like calprotectin; and thereby evaluating the suitability of current plasma EVs analysis techniques for identifying new biomarkers of HIV-1 immune activation. Multiple analysis have been performed on HIV-1 infected cell lines, plasma from HIV-1 patients, or plasma from HIV-negative individuals spiked with HIV-1. Commercial kits, the differential centrifugation and density or velocity gradients to precipitate and separate HIV, EVs, and proteins such as calprotectin, have been used. EVs, virions, and contaminating proteins were characterized using Western blot, ELISA, RT-PCR, hydrodynamic size measurement, and enzymatic assay. Conversely to iodixanol density or velocity gradient, protein and virions co-sedimented in the same fractions of the sucrose density gradient than AChE-positive EVs. Iodixanol velocity gradient provided the optimal separation of EVs from viruses and free proteins in culture supernatants and plasma samples from a person living with HIV (PLWH) or a control and revealed a new population of large EVs enriched in microRNA miR-155 and mitochondrial DNA. Although EVs and their contents provide helpful information about several key events in HIV-1 pathogenesis, their purification and extensive characterization by velocity gradient must be investigated thoroughly before further use as biomarkers. By revealing a new population of EVs enriched in miR-155 and mitochondrial DNA, this study paves a way to increase our understanding of HIV-1 pathogenesis.

Characterization of Cloned Mitochondrial DNA from the Teleost Fundulus heteroclitus and its Usefulness as an Interspecies Hybridization Probe

1986 ◽  
Vol 43 (10) ◽  
pp. 1866-1872 ◽  
Author(s):  
Lucia Irene González-Villaseñor ◽  
Amanda M. Burkhoff ◽  
Víctor Corces ◽  
Dennis A. Powers
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Fundulus Heteroclitus ◽  
Staining Technique ◽  
Hybridization Probe ◽  
Cross Hybridization ◽  
Restriction Patterns ◽  
Ethidium Bromide Staining ◽  
Dna Restriction Fragments ◽  
Dna Restriction

Analysis of mitochondrial DNA endonuclease restriction patterns is a powerful tool for studying related species and variation within species. The ethidium bromide staining technique has limited the number of digestions of mitochondrial DNA per individual. Because 32P-end-labeling also imposes severe limitations, we have resorted to cloning the fish (Fundulus heteroclitus) mitochondrial genome in the lambda replacement vector EMBL-3. The clone was used as a radioactive probe via Southern blotting to detect mitochondrial DNA restriction fragments obtained by multiple restriction endonuclease digestions from small amounts of tissue. This technique offers much greater sensitivity than ethidium bromide staining. Moreover, it eliminates the expense and time to obtain highly purified mitochondrial DNA for the 32P-end-labeling procedure. It is also useful when the mtDNA is prepared from frozen tissue which has been a problem with the 32P-end-labeling technique. Because the cloned mitochondrial DNA has a high degree of cross-hybridization with the mitochondrial DNA of certain other fishes, it can be used to probe the mitochondrial DNA restriction patterns of a variety of fish species. However, its usefulness is restricted by the degree of relatedness to the species being cloned.

The effects of spectinomycin and ethidium bromide on the synthesis of organelle rRNA and on ultrastructure in Ochromonas danica

1980 ◽  
Vol 43 (1) ◽  
pp. 119-136
Author(s):  
H. Smith-Johannsen ◽  
D. Fromson ◽  
S.P. Gibbs
Keyword(s):  
Electron Microscopy ◽  
Ethidium Bromide ◽  
Ribosomal Proteins ◽  
Specific Inhibitor ◽  
Chloroplast Ultrastructure ◽  
Normal Amount ◽  
Chloroplast Membranes ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Rrna ◽  
Ochromonas Danica

The effects of 24-h exposure to spectinomycin (100 microgram/ml) and ethidium bromide (1 microgram/ml) on the accumulation of chloroplast and mitochondrial rRNAs and on organelle ultrastructure were studied in greening cells of Ochromonas danica. Cells treated with ethidium bromide for 24 h divide at the same rate as controls but contain less than one third the normal amount of mitochondrial rRNA. Ultrastructural observations showed that these cells contain only 10% the number of mitochondrial ribosomes found in controls as well as fewer mitochondrial cristae. Ethidium bromide has no effect on chloroplast ultrastructure in Ochromonas. Greening cells treated with spectinomycin grow at close to control rates but contain 30–40% less chloroplast rRNA than do controls. Electron microscopy showed that spectinomycin disrupts the organization of chloroplast membranes and reduces the number of chloroplast ribosomes by 30%. Under these conditions, spectinomycin has no effect on mitochondrial rRNA or ultrastructure. Since spectinomycin is a specific inhibitor of translation on 70S ribosomes, these results are consistent with the possibility that at least some chloroplast ribosomal proteins are synthesized in the chloroplast of Ochromonas.

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