Preferential delection of a specific region of mitochondrial DNA in Saccharomyces cerevisiae by ethidium bromide and 3-carbethoxy-psoralen

1978 ◽  
Vol 162 (2) ◽  
pp. 191-201 ◽  
Author(s):  
H. Fukuhara ◽  
E. Moustacchi ◽  
M. Wesolowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Specific Region

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.

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.

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.

scholarly journals Mitochondrial DNA duplication, recombination, and introgression during interspecific hybridization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Silvia Bágeľová Poláková ◽  
Žaneta Lichtner ◽  
Tomáš Szemes ◽  
Martina Smolejová ◽  
Pavol Sulo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Interspecific Hybridization ◽  
Mobile Elements ◽  
Variable Length ◽  
Mitochondrial Genomes ◽  
Free Standing ◽  
Saccharomyces Paradoxus ◽  
Mtdna Recombination

AbstractmtDNA recombination events in yeasts are known, but altered mitochondrial genomes were not completed. Therefore, we analyzed recombined mtDNAs in six Saccharomyces cerevisiae × Saccharomyces paradoxus hybrids in detail. Assembled molecules contain mostly segments with variable length introgressed to other mtDNA. All recombination sites are in the vicinity of the mobile elements, introns in cox1, cob genes and free standing ORF1, ORF4. The transplaced regions involve co-converted proximal exon regions. Thus, these selfish elements are beneficial to the host if the mother molecule is challenged with another molecule for transmission to the progeny. They trigger mtDNA recombination ensuring the transfer of adjacent regions, into the progeny of recombinant molecules. The recombination of the large segments may result in mitotically stable duplication of several genes.

scholarly journals Maintenance of Mitochondrial Morphology Is Linked to Maintenance of the Mitochondrial Genome in Saccharomyces cerevisiae

Genetics ◽  
2002 ◽  
Vol 162 (3) ◽  
pp. 1147-1156 ◽  
Author(s):  
Theodor Hanekamp ◽  
Mary K Thorsness ◽  
Indrani Rebbapragada ◽  
Elizabeth M Fisher ◽  
Corrine Seebart ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Mitochondrial Dna ◽  
Carbon Sources ◽  
Mitochondrial Morphology ◽  
Yeast Saccharomyces Cerevisiae ◽  
Slow Growth Rate ◽  
Dna Migration ◽  
Mitochondrial Dna Stability ◽  
Extragenic Suppressors

Abstract In the yeast Saccharomyces cerevisiae, certain mutant alleles of YME4, YME6, and MDM10 cause an increased rate of mitochondrial DNA migration to the nucleus, carbon-source-dependent alterations in mitochondrial morphology, and increased rates of mitochondrial DNA loss. While single mutants grow on media requiring mitochondrial respiration, any pairwise combination of these mutations causes a respiratory-deficient phenotype. This double-mutant phenotype allowed cloning of YME6, which is identical to MMM1 and encodes an outer mitochondrial membrane protein essential for maintaining normal mitochondrial morphology. Yeast strains bearing null mutations of MMM1 have altered mitochondrial morphology and a slow growth rate on all carbon sources and quantitatively lack mitochondrial DNA. Extragenic suppressors of MMM1 deletion mutants partially restore mitochondrial morphology to the wild-type state and have a corresponding increase in growth rate and mitochondrial DNA stability. A dominant suppressor also suppresses the phenotypes caused by a point mutation in MMM1, as well as by specific mutations in YME4 and MDM10.

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.

scholarly journals Saccharomyces cerevisiae contains an RNase MRP that cleaves at a conserved mitochondrial RNA sequence implicated in replication priming.

1992 ◽  
Vol 12 (6) ◽  
pp. 2561-2569 ◽  
Author(s):  
L L Stohl ◽  
D A Clayton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Dna Replication ◽  
Rna Processing ◽  
Mitochondrial Rna ◽  
Rna Sequence ◽  
Rnase Mrp ◽  
Processing Activity ◽  
Yeast Mitochondrial Dna ◽  
Human And Mouse

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

scholarly journals Expression of the Saccharomyces cerevisiae Gene YME1 in the Petite-Negative Yeast Schizosaccharomyces pombe Converts It to Petite-Positive

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 147-154 ◽  
Author(s):  
Douglas J Kominsky ◽  
Peter E Thorsness
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Dna ◽  
Schizosaccharomyces Pombe ◽  
Atp Synthase ◽  
Kluyveromyces Lactis ◽  
Blot Hybridization ◽  
Inner Mitochondrial Membrane ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitochondrial Atp Synthase ◽  
Petite Negative Yeast

Abstract Organisms that can grow without mitochondrial DNA are referred to as “petite-positive” and those that are inviable in the absence of mitochondrial DNA are termed “petite-negative.” The petite-positive yeast Saccharomyces cerevisiae can be converted to a petite-negative yeast by inactivation of Yme1p, an ATP- and metal-dependent protease associated with the inner mitochondrial membrane. Suppression of this yme1 phenotype can occur by virtue of dominant mutations in the α- and γ-subunits of mitochondrial ATP synthase. These mutations are similar or identical to those occurring in the same subunits of the same enzyme that converts the petite-negative yeast Kluyveromyces lactis to petite-positive. Expression of YME1 in the petite-negative yeast Schizosaccharomyces pombe converts this yeast to petite-positive. No sequence closely related to YME1 was found by DNA-blot hybridization to S. pombe or K. lactis genomic DNA, and no antigenically related proteins were found in mitochondrial extracts of S. pombe probed with antisera directed against Yme1p. Mutations that block the formation of the F1 component of mitochondrial ATP synthase are also petite-negative. Thus, the F1 complex has an essential activity in cells lacking mitochondrial DNA and Yme1p can mediate that activity, even in heterologous systems.

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