Inhibition of mTOR Prevents ROS Production Initiated by Ethidium Bromide-Induced Mitochondrial DNA Depletion

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.

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Faculty Opinions recommendation of Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1086992.539949 ◽

2007 ◽

Author(s):

Michio Hirano

Keyword(s):

Mitochondrial Dna ◽

Ribonucleotide Reductase ◽

Mitochondrial Dna Depletion

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Faculty Opinions recommendation of MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12481.469965 ◽

2006 ◽

Author(s):

Michio Hirano

Keyword(s):

Mitochondrial Dna ◽

Membrane Protein ◽

Mitochondrial Membrane ◽

Inner Mitochondrial Membrane ◽

Mitochondrial Dna Depletion ◽

Mitochondrial Membrane Protein

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

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa119 ◽

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.

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

Canadian Journal of Fisheries and Aquatic Sciences ◽

10.1139/f86-231 ◽

1986 ◽

Vol 43 (10) ◽

pp. 1866-1872 ◽

Cited By ~ 10

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.

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