Directed Alteration of Saccharomyces cerevisiae Mitochondrial DNA by Biolistic Transformation and Homologous 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.

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

Genetics ◽

10.1093/genetics/162.3.1147 ◽

2002 ◽

Vol 162 (3) ◽

pp. 1147-1156 ◽

Cited By ~ 1

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.

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Assembly of the mitochondrial membrane system. Complete restriction map of the cytochrome b region of mitochondrial DNA in Saccharomyces cerevisiae D273-10B.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43466-7 ◽

1980 ◽

Vol 255 (20) ◽

pp. 9821-9827

Author(s):

F.G. Nobrega ◽

A. Tzagoloff

Keyword(s):

Saccharomyces Cerevisiae ◽

Mitochondrial Dna ◽

Cytochrome B ◽

Mitochondrial Membrane ◽

Membrane System ◽

Restriction Map

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A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5130-5143.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5130-5143 ◽

Cited By ~ 32

Author(s):

Christine Soustelle ◽

Laurence Vernis ◽

Karine Fréon ◽

Anne Reynaud-Angelin ◽

Roland Chanet ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Homologous Recombination ◽

Nonhomologous End Joining ◽

End Joining ◽

Recombination Mechanism ◽

Growth Defects ◽

Protein Conjugates ◽

Synthetically Lethal ◽

Mutant Cells ◽

Insight Into

ABSTRACT The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

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Effect of the expression of BRCA2 on spontaneous homologous recombination and DNA damage-induced nuclear foci in Saccharomyces cerevisiae

Mutagenesis ◽

10.1093/mutage/ges069 ◽

2013 ◽

Vol 28 (2) ◽

pp. 187-195 ◽

Cited By ~ 9

Author(s):

L. Spugnesi ◽

C. Balia ◽

A. Collavoli ◽

E. Falaschi ◽

V. Quercioli ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Homologous Recombination ◽

Nuclear Foci

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

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2561 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2561-2569 ◽

Cited By ~ 37

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.

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

Genetics ◽

10.1093/genetics/154.1.147 ◽

2000 ◽

Vol 154 (1) ◽

pp. 147-154 ◽

Cited By ~ 1

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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