Chromosomal DNA patterns and mitochondrial DNA polymorphism as tools for identification of enological strains of Saccharomyces cerevisiae

Summary Chromosomal DNA patterns using the transverse alternating field electrophoresis technique and mitochondrial DNA restriction profiles have been achieved for 22 enological strains of Saccharomyces cerevisiae. Both methods have evidenced a marked polymorphism of these strains. Twenty different karyotypes and 17 mitochondrial DNA banding patterns have been observed. Only three strains originating from the same vineyard could not be differentiated by either of the two methods. The polymorphism observed at the chromosomal and mitochondrial levels makes the techniques investigated powerful tools for identification and control of industrial strains.Offprint requests to: J.-N. Hallet     

Evidence for Mitochondrial DNA Polymorphism and Uniparental Inheritance in the Cellular Slime Mold Polysphondylium Pallidum: Effect of Intraspecies Mating on Mitochondrial DNA Transmission

Restriction fragment length polymorphisms (RFLPs) were used as markers to monitor mitochondrial inheritance in the cellular slime mold, Polysphondylium pallidum. When two opposite mating types (mat1 and mat2) of closely related strains were crossed, all the haploid progeny regardless of mating type inherited their mitochondrial DNA from the mat2 parent only. When opposite mating types from more distantly related strains were crossed, most of the progeny also inherited their mitochondrial DNA from the mat2 parent, but some inherited their mitochondrial DNA from the mat1 parent. In both cases however, the transmission of mitochondrial DNA was uniparental, since in every individual progeny only one type of mitochondrial DNA exists. Moreover, in crosses involving more distantly related strains all the progeny of a single macrocyst were shown to contain the same type of mitochondrial DNA. These findings are discussed in regard to mechanisms of transmission and the possible involvement of nuclear genes in the control of transmission of mitochondrial DNA in Polysphondylium.     

香菇野生菌株线粒体DNA多态性

利用PCR技术扩增了我国 7个省的 2 0个野生香菇菌株的线粒体DNA的 2个小区段 ,利用限制性酶切技术研究这 2个片段长度多型性 (RFLPs) ,结果显示 ,2 0个野生香菇菌株间的线粒体DNA在研究的区段上分为 2大类型 ,相似性为 70 % ,与栽培种相比存在较大的遗传差异。     

Replication of bromodeoxyuridylate-substituted mitochondrial DNA in yeast.

The DNA of several strains of Saccharomyces cerevisiae was labeled by growing the culture in medium supplemented with thymidylate and bromodeoxyuridylate. It was thus possible to follow the course of mitochondrial DNA replication in density shift experiments by determining the buoyant density distribution of unreplicated and replicated DNAs in analytical CsCl gradients. DNA replication was followed for three generations after transfer of cultures from light medium to heavy medium and heavy medium to light medium. Under both conditions, the density shifts observed for mitochondrial DNA were those expected for semiconservative, nondispersive replication. This was further confirmed by analysis of the buoyant density of alkali-denatured hybrid mitochondrial DNA. With this method, no significant recombination between replicated and unreplicated DNA was detected after three generations of growth.     

Mutations in the Mitochondrial Atp Synthase Gamma Subunit Suppress a Slow-Growth Phenotype of Yme1 Yeast Lacking Mitochondrial DNA

In Saccharomyces cerevisiae, inactivation of the nuclear gene YME1 causes several phenotypes associated with impairment of mitochondrial function. In addition to deficiencies in mitochondrial compartment integrity and respiratory growth, yme1 mutants grow extremely slowly in the absence of mitochondrial DNA. We have identified two genetic loci that, when mutated, act as dominant suppressors of the slow-growth phenotype of yme1 strains lacking mitochondrial DNA. These mutations only suppressed the slow-growth phenotype of yme1 strains lacking mitochondrial DNA and had no effect on other phenotypes associated with yme1 mutations. One allele of one linkage group had a collateral respiratory deficient phenotype that allowed the isolation of the wild-type gene. This suppressing mutation was in ATP3, a gene that encodes the gamma subunit of the mitochondrial ATP synthase. Recovery of two of the suppressing ATP3 alleles and subsequent sequence analysis placed the suppressing mutations at strictly conserved residues near the C terminus of Atp3p. Deletion of the ATP3 genomic locus resulted in an inability to utilize nonfermentable carbon sources. atp3 deletion strains lacking mitochondrial DNA grew slowly on glucose media but were not as compromised for growth as yme1 yeast lacking mitochondrial DNA.     

Unusual type of mitochondrial DNA in mice lacking a maternally transmitted antigen

Mice that lack a maternally transmitted antigen (Mta) on the cell surface share a distinctive type of mitochondrial DNA. This is evident from restriction analyses of mitochondrial DNAs from 25 strains of mice whose antigenic state is known. One hundred sixty-eight cleavage sites have been mapped in the mitochondrial DNA of Mta- mice. Detailed maps for the 8 other types of mitochondrial DNA detected in the survey have also been prepared. The Mta- mice are estimated to differ from those expressing the antigen by 108 to 141 base substitutions at widely scattered points in the mitochondrial genome.     

Flocculation in Saccharomyces cerevisiae and mitochondrial DNA structure

Summary Mitochondrial mutants of indstrial yeast strains with different flocculation efficiencies were assayed for involvement of mitochondrial DNA (mtDNA) in flocculation. Most of the mutants exhibited a decreased flocculation rate in comparison to that of the wild strains. The mtDNA of a moderately flocculating wild strain was characterized by restriction enzyme analysis and by the localization of several mitochondrial genes. This molecular analysis of mitochondrial mutants revealed two areas of mtDNA involvement in flocculation, namely a region of the subunit 9 of the ATPase gene (oli 1) and a region of the subunit 3 of the cytochrome-c-oxidase gene (oxi 2).     

Nuclear and mitochondr1al DNA synthesis during yeast sporulation

Nuclear and mitochondrial DNA synthesis during sporulation of Saccharomyces cerevisiae has been studied in a wild-type (aα) strain and 3 sporulation deficient strains. We find that in a strain carrying a dominant mutation which prevents sporulation, nuclear DNA synthesis is initiated but not completed; mitochondrial DNA synthesis, on the other hand, does take place. In aa and αα diploids no initiation of nuclear DNA synthesis is seen to occur, and only a very low level of mitochondrial DNA synthesis is observed. We conclude that mitochondrial DNA synthesis in sporulation medium is uncoupled from nuclear DNA synthesis. In addition, the steps at which the sporulation process is arrested in aa and αα cells and in the dominant mutant can be ordered in time as being before and after the initiation of nuclear DNA synthesis.     

香菇栽培种线粒体DNA和核糖体DNA多态性研究初探

本文应用RFLP技术研究了10个香菇主栽品种的线粒体DNA（mtDNA）和核糖体DNA（rDNA）的部分小区段,利用PCR技术扩增了rDNA5．8＋ITS区段及mtDNA的小区段,分析这些片段的限制性酸切图谱,并进行菌株间的遗传相似系数的估算。结果显示：菌株间的rDNA在5．8＋ITS区段差异很小,表明同一种内菌株间的rDNA具有相对的遗传稳定性;不同菌株间未检出mtDNA的差异,表明菌株间在所研究的区段具有很高的遗传相似性。     

Biogenesis of mitochondria

Summary The proportion of total cell DNA which is mitochondrial DNA was measured in haploid, diploid and tetraploid strains of S. cerevisiae grown under a standard set of conditions. For all strains tested the mitochondrial DNA level was in the range 16%–25% of total cell DNA. Repeated measurements of the cellular level of mitochondrial DNA in two haploid strains showed that these strains have measurably different cellular mitochondrial DNA levels (17% and 24% of total DNA, respectively) under our conditions. These two grande strains were used to investigate the role of the mitochondrial and nuclear genomes in the regulation of the mitochondrial DNA level. We have shown by genetic analysis that the difference between these two strains is determined by at least two nuclear genes. The mitochondrial genome is not involved in the regulation of cellular mitochondrial DNA levels.A number of purified petite clones derived from independent spontaneous petite isolates of the grande strain which contained 24% mitochondrial DNA were also studied. The mitochondrial DNA levels in all but one of these petites fell in the range 20–25% of total cell DNA. From these results we conclude that, in general, the mitochondrial DNA level in petite strains is controlled by the same mechanism as operates in grande strains.We propose a general model for the control of the cellular mitochondrial DNA level, in which the amount of mitochondrial DNA per cell is determined by regulation of the number of mitochondrial DNA molecules per cell. This regulation is mediated through the availability of a set of nuclear coded components, possibly a mitochondrial membrane site, which are required for the replication of mitochondrial DNA.     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

M13 phage DNA as a universal marker for DNA fingerprinting of animals, plants and microorganisms

Hypervariable polymorphic patterns were detected with M13 phage DNA as a probe in genomic DNA of organisms belonging to different taxonomic groups including animals (vertebrates and invertebrates), plants and microorganisms. Individual-specific restriction pattern analysis (DNA fingerprinting) with this probe proved to be useful for individual identification, analysis of somatic stability and paternity testing in man. The nuclear type of inheritance indicates that the hypervariable DNA regions in question are located in the chromosomes, not in the mitochondrial DNA. The data obtained also demonstrate a potential range of M13 DNA applications as a probe for DNA fingerprinting of animals, plants and microorganisms, particularly for the determination of inbred lines, identification of bacterial strains and establishing stock, variety and strain distinctions.     

Developing a genetic approach to investigate the mechanism of mitochondrial competence for DNA import

Mitochondrial gene products are essential for the viability of eukaryote obligate aerobes. Consequently, mutations of the mitochondrial genome cause severe diseases in man and generate traits widely used in plant breeding. Pathogenic mutations can often be identified but direct genetic rescue remains impossible because mitochondrial transformation is still to be achieved in higher eukaryotes. Along this line, it has been shown that isolated plant and mammalian mitochondria are naturally competent for importing linear DNA. However, it has proven difficult to understand how such large polyanions cross the mitochondrial membranes. The genetic tractability of Saccharomyces cerevisae could be a powerful tool to unravel this molecular mechanism. Here we show that isolated S. cerevisiae mitochondria can import linear DNA in a process sharing similar characteristics to plant and mammalian mitochondria. Based on biochemical data, translocation through the outer membrane is believed to be mediated by voltage-dependent anion channel (VDAC) isoforms in higher eukaryotes. Both confirming this hypothesis and validating the yeast model, we illustrate that mitochondria from S. cerevisiae strains deleted for the VDAC-1 or VDAC-2 gene are severely compromised in DNA import. The prospect is now open to screen further mutant yeast strains to identify the elusive inner membrane DNA transporter.     

Complete DNA sequences of the mitochondrial genomes of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis: insight into the evolution of linear DNA genomes from mitochondrial telomere mutants

We determined complete mitochondrial DNA sequences of the two yeast species, Candida orthopsilosis and Candida metapsilosis, and compared them with the linear mitochondrial genome of their close relative, C.parapsilosis. Mitochondria of all the three species harbor compact genomes encoding the same set of genes arranged in the identical order. Differences in the length of these genomes result mainly from the presence/absence of introns. Multiple alterations were identified also in the sequences of the ribosomal and transfer RNAs, and proteins. However, the most striking feature of C.orthopsilosis and C.metapsilosis is the existence of strains differing in the molecular form of the mitochondrial genome (circular-mapping versus linear). Their analysis opens a unique window for understanding the role of mitochondrial telomeres in the stability and evolution of molecular architecture of the genome. Our results indicate that the circular-mapping mitochondrial genome derived from the linear form by intramolecular end-to-end fusions. Moreover, we suggest that the linear mitochondrial genome evolved from a circular-mapping form present in a common ancestor of the three species and, at the same time, the emergence of mitochondrial telomeres enabled the formation of linear monomeric DNA forms. In addition, comparison of isogenic C.metapsilosis strains differing in the form of the organellar genome suggests a possibility that, under some circumstances, the linearity and/or the presence of telomeres provide a competitive advantage over a circular-mapping mitochondrial genome.     

Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA

Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.     

Loss of the Mitochondrial Nucleoid Protein, Abf2p, Destabilizes Repetitive DNA in the Yeast Mitochondrial Genome

Loss of Abf2p, an abundant mitochondrial nucleoid-associated protein, results in increased mitochondrial frameshifts and direct-repeat mediated deletions but has no effect on the rate of mitochondrial point mutations. The instability of repeated sequences in this strain may be linked to the loss of mitochondrial DNA in abf2-Δ strains.     

Global Genetic Determinants of Mitochondrial DNA Copy Number

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role.     

A fluorescent-cytochemical method for selective detection of yeast mitochondrial DNA

Yeast mitochondrial DNA (mDNA) can selectively be detected using a specific dye (DAPI). Nuclear DNA (nDNA) was stained along with mDNA only in three out of the fifteen studied yeast strains. Visualisation with a luminescent microscope showed that mDNA content varied among different yeast species as well as the size and shape of fluorescent mitochondrial nuclei. Intensive nDNA fluorescence was detected when a fixed specimen was treated with DAPI. Under these conditions, mDNA was revealed only in yeast cells with its high content. The process of fixation was shown to interfere with the integrity of mitochondria. It is also possible that the structure of DNA was modified to affect its interaction with the dye and thus the level of fluorescence. The developed technique of selective mDNA staining and the experimental results make it possible to gain a more accurate quantitative information about DNA content at the cellular level using cyto- and spectrofluorimetric techniques. This study involves important aspects pertinent to the dye interaction with yeast nuclear and mitochondrial DNA, both native and subjected to different fixation procedures.     

洞庭青鲫与其他六个鲫鱼品系线粒体DNA控制区的比较分析

运用PCR扩增、克隆、测序等技术,在获得洞庭青鲫和彭泽鲫线粒体DNA控制区全序列的基础上,对洞庭青鲫、彭泽鲫、普通鲫、红鲫、白鲫、A系和D系银鲫等7个鲫鱼品系线粒体DNA控制区的碱基组成、变异情况、序列结构和系统进化进行了比较分析。结果表明,7个鲫鱼品系线粒体DNA控制区碱基的平均组成为A:(32.7±0.16)%,C:(20.4±0.37)%,G:(14.1±0.08)%,T:(32.8±0.36)%,序列分歧率为0—5.7%,其中红鲫和白鲫的分歧率最大(5.7%),彭泽鲫、A系和D系银鲫之间最小(0)。洞庭青鲫与白鲫的分歧率为5.6%,与彭泽鲫、A系和D系银鲫为2.2%,与红鲫和普通鲫分别为0.8%和0.7%。对比哺乳动物线粒体DNA控制区结构,并参照其他鱼类序列,将7个鲫鱼品系的控制区分为终止序列区、中央保守区和保守序列区三个区域。同时识别了7个鲫鱼品系中一系列保守序列,并给出了它们的一般形式。序列差异和系统进化分析表明,在7个鲫鱼品系中,洞庭青鲫与普通鲫和红鲫的亲缘关系最近,与彭泽鲫、A系和D系银鲫亲缘关系次之,与白鲫的亲缘关系最远,而彭泽鲫、A系和D系银鲫三者在线粒体DNA控制区的相似性和分歧率上则表现为是同一个鲫鱼品系。