Rapid Isolation of Mitochondrial DNA-Depleted Mammalian Cells by Ethidium Bromide and Dideoxycytidine Treatments

2014 ◽  
Vol 57 (3) ◽  
pp. 259-265 ◽  
Author(s):  
Young Geol Yoon ◽  
Yoo Jin Oh ◽  
Young Hyun Yoo
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Mammalian Cells ◽  
Rapid Isolation

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.

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.

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.

Limitations of Allotopic Expression of Mitochondrial Genes in Mammalian Cells

Genetics ◽  
2003 ◽  
Vol 165 (2) ◽  
pp. 707-720
Author(s):  
Jose Oca-Cossio ◽  
Lesley Kenyon ◽  
Huiling Hao ◽  
Carlos T Moraes
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells ◽  
Mitochondrial Genes ◽  
Terminal Segment ◽  
Universal Genetic Code ◽  
Apocytochrome B ◽  
Dna Mutations ◽  
C Terminus ◽  
Cytoplasmic Compartment ◽  
Allotopic Expression

Abstract The possibility of expressing mitochondrial DNA-coded genes in the nuclear-cytoplasmic compartment provides an attractive system for genetic treatment of mitochondrial disorders associated with mitochondrial DNA mutations. In theory, by recoding mitochondrial genes to adapt them to the universal genetic code and by adding a DNA sequence coding for a mitochondrial-targeting sequence, one could achieve correct localization of the gene product. Such transfer has occurred in nature, and certain species of algae and plants express a number of polypeptides that are commonly coded by mtDNA in the nuclear-cytoplasmic compartment. In the present study, allotopic expression of three different mtDNA-coded polypeptides (ATPase8, apocytochrome b, and ND4) into COS-7 and HeLa cells was analyzed. Among these, only ATPase8 was correctly expressed and localized to mitochondria. The full-length, as well as truncated forms, of apocytochrome b and ND4 decorated the periphery of mitochondria, but also aggregated in fiber-like structures containing tubulin and in some cases also vimentin. The addition of a hydrophilic tail (EGFP) to the C terminus of these polypeptides did not change their localization. Overexpression of molecular chaperones also did not have a significant effect in preventing aggregations. Allotopic expression of apocytochrome b and ND4 induced a loss of mitochondrial membrane potential in transfected cells, which can lead to cell death. Our observations suggest that only a subset of mitochondrial genes can be replaced allotopically. Analyses of the hydrophobic patterns of different polypeptides suggest that hydrophobicity of the N-terminal segment is the main determinant for the importability of peptides into mammalian mitochondria.

Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells

1985 ◽  
Vol 160 (2) ◽  
pp. 387-395 ◽  
Author(s):  
Jun-Ichi Hayashi ◽  
Yusaku Tagashira ◽  
Michihiro C. Yoshida
Keyword(s):  
Mitochondrial Dna ◽  
Mammalian Cells

SACCHAROMYCES CEREVISIAE PETITE MITOCHONDRIAL DNA OF SUPPRESSIVE AND NEUTRAL HAPLOIDS AND OF [rho−] DIPLOIDS OBTAINED FROM CROSSING [rho+] TO A NEUTRAL PETITE

1975 ◽  
Vol 17 (3) ◽  
pp. 381-389 ◽  
Author(s):  
A. J. Robertson ◽  
N. T. Bech-Hansen ◽  
G. H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Buoyant Density ◽  
Unusual Property ◽  
Mating Reaction

An unusual property of GR25a [rho+] was the production of 20 to 30 percent [rho−] zygote colonies when crossed to a tester strain lacking mitochondrial DNA. Spontaneous [rho−] isolates of GR25a [rho+] were observed to be highly suppressive and to contain mitochondrial DNA of a parental buoyant density (1.685 g/cm3). Three ethidium bromide induced neutral petites of GR25a [rho+] did not have detectable mitochondrial DNA and were neutral in crosses to [rho+] strains. Seven [rho−] zygote colony isolates obtained from crossing GR25a [rho+] to a neutral petite were shown to contain abnormal mitochondrial DNA. Six zygote colony isolates had mitochondrial DNA of a buoyant density less than, or equal to, GR25a (1.682–1.685 g/cm3), whereas one isolate had a buoyant density greater than GR25a (1.688 g/cm3). It was suggested that abnormal mitochondrial DNA is generated during the mating reaction.

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts

1985 ◽  
Vol 5 (5) ◽  
pp. 1163-1169
Author(s):  
P Desjardins ◽  
E Frost ◽  
R Morais
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Chicken Embryo ◽  
Blot Hybridization ◽  
Cell Populations ◽  
Free Medium ◽  
Reassociation Kinetics ◽  
Embryo Fibroblasts ◽  
Drug Free ◽  
Chicken Embryo Fibroblasts

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

SV40-transformed hamster cells resistant to 100–250 microgram/ml of ethidium bromide

1978 ◽  
Vol 33 (1) ◽  
pp. 157-169
Author(s):  
G. Wolf ◽  
L. Tejmar ◽  
S. Borell ◽  
W. Klietman
Keyword(s):  
Mitochondrial Dna ◽  
Cell Lines ◽  
Ethidium Bromide ◽  
Gel Electrophoresis ◽  
Mitochondrial Proteins ◽  
Wild Type ◽  
Mitochondrial Ultrastructure ◽  
Gold Hamsters ◽  
Resistant Cells

SV40-transformed hamster cells were selected for resistance to ethidium bromide (EB). Several cell lines were established, which grew in the presence of up to 250 microgram/ml EB. The EB resistance is genetically stable. The cloned resistant cells show no difference in morphology, with the exception of the mitochondrial ultrastructure, which exhibits condensed cristae formation. The tumorigenicity of these cells in Syrian gold hamsters is considerably reduced. Incorporation of radioactive labelled thymidine into mitochondrial DNA is not influenced by the presence of the drug. Gel electrophoresis with mitochondrial proteins from wild-type and resistant cells reveals significantly different patterns. The mechanism of EB resistance is discussed.

Ethidium bromide as a vital probe of mitochondrial DNA in carcinoma cells

2009 ◽  
Vol 45 (14) ◽  
pp. 2588-2597 ◽  
Author(s):  
Anna Maria Villa ◽  
Silvia Maria Doglia
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Carcinoma Cells
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