The organization of genes in yeast mitochondrial DNA

Summary We have fractionated fragments of yeast mtDNA, obtained with restriction endonucleases, on poly(U)-Sephadex columns using the procedure of Flavell and Van den Berg (FEBS Letters (1975) 58, 90–93). The poly(U) forms a triple helix with (dA·dT) clusters in duplex DNA and fractionates DNA fragments on the basis of the length and number of clusters contained in them.mtDNA fragments obtained with endonucleases PstI, BamHI, HindII, HindII+III, EcoRI, HapII and HhaI were separated by poly(U)-Sephadex in three groups: fragments not retained by the column in 2M LiCl, fragments partially retained and fragments (nearly) completely bound in 2 M LiCl and only eluted by 0.1 M LiCl. The separation obtained is adequate for analytical fractionation of fragments and it can be used for the preparative isolation of firmly-bound fragments.In mtDNA digests made with endonuclease HapII, which gives about 70 separable fragments under our conditions, only about 10% of the fragments were firmly bound to poly(U)-Sephadex. This shows that the number of (dA·dT) clusters long enough to result in binding is limited in yeast mtDNA and its suggests that large fragments are bound by only one or a few clusters.Corresponding segments of the physical map of the mtDNAs from Saccharomyces carlsbergensis and Saccharomyces cerevisiae strains JS1-3D and KL14-4A were bound to the column, showing that the (dA·dT) clusters responsible for binding are conserved in the evolution of mtDNA. However, one 3,000 bp insert, only present on KL14-4A mtDNA, causes the loss of a binding site, another long insert introduces a new binding site.Fragments firmly bound to the columns are clustered in one quadrant of the physical map of these three mtDNAs. This quadrant also contains the large insertions present in KL14-4A mtDNA and absent from S. carlsbergensis mtDNA. The possible relation between (dA·dT) clusters and insertions is discussed.Abbreviation bp base pairs     

Recombination in yeast mitochondrial DNA

When haploid yeast strains containing mitochondrial DNAs (mtDNAs) of different buoyant densities are mated, the resulting zygotes contain a mixed population of mitochondria and mitochondrial DNAs. During vegetative growth of diploid cells formed from such a cross between a petite strain with mtDNA of density 1.677 g cm^?3 and a respiratory competent strain with mtDNA of density 1.684 g cm^?3, mtDNAs with intermediate buoyant densities are obtained. Virtually all newly synthesized mtDNA in diploid ρ^? progeny has the intermediate buoyant density. Therefore, within 2 generations of growth of the diploid cells, the intermediate buoyant density species predominate. In crosses between a respiratory competent strain and other petite strains with different values of genetic suppressiveness, it was found that the amount of recombination yielding mtDNAs of intermediate buoyant densities roughly parallels the degree of suppressiveness. Individual clones of respiratory deficient cells from such crosses were also isolated to confirm that stable mtDNAs with intermediate buoyant densities were obtained. Thus, it is apparent that some form of recombination takes place within the mtDNAs of yeast cells that results in stable mtDNA species.     

Regulation of yeast mitochondrial DNA synthesis

Summary A single recessive nuclear gene mutation has been isolated from strain 123.1 C of Saccharomyces cerevisiae which is conditionally deficient in mitochondrial DNA metabolism and has been termed tpi. Growth of this mutant strain in media containing galactose at 36°C causes a reduction of mitochondrial DNA synthesis as analyzed by incorporation of radioactive adenine into the mitochondrial DNA. These cells continue to grow and divide producing petite cells which are neutral and have been found to lack mitochondrial DNA as measured by radioactive incorporation of ³H-adenine into the mitochondrial DNA in the presence of cycloheximide at the permissive temperature. The rate of mitochondrial DNA synthesis of the mutant strain grown at the restrictive temperature in dextrose or glycerol containing media was found to be greatly reduced following two hours of exposure to the restrictive temperature. In addition, the action of this mutant gene has been found to be independent of the respiratory capacity of the mutant strain.     

Regulation of yeast mitochondrial DNA synthesis

Summary The expression and stability of Escherichia coli F-primes in Proteus mirabilis is examined. It is possible to consecutively introduce, and stably maintain, the DNA of several E. coli F-primes in P. mirabilis in the absence of selective pressure for all or some of the plasmids. Additionally, we can recover more than one F-prime from certain P. mirabilis recipient strains which carry DNA derived from several independent matings with E. coli F-prime donors.     

A yeast with linear molecules of mitochondrial DNA

Summary Mitochondrial DNA from the yeast strain SR23, tentatively allocated to the species, Candida rhagii, consists of linear molecules 30 kb long. This has been demonstrated by restriction analysis and selective radioactive labelling of terminal restriction fragments. Preliminary sequence analysis indicated that the two ends of the molecule are formed by inverted repeats. The arrangement of several genes in the mitochondrial genome of C. rhagii SR23 was established by specific hybridisation with probes prepared from mitochondrial DNA of Saccharomyces cerevisiae. The arrangement is unique, with genes coding for the two ribosomal RNAs placed widely apart. Intron(s) may be present in the gene coding for cytochrome b.     

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.     

Segments of mitochondrial DNA of yeast carrying antibiotic resistances

Summary Mitochondrial DNA was isolated from an oligomycin-resistant petite mutant of yeast, Saccharomyces cerevisiae. It had repeated sequences of 3600 base pairs. This segment was about one twentieth of the whole mtDNA of wild type yeast, which had a size of 74 kilo base pairs.This segment of mtDNA had one cleavage site for a restriction endonuclease, Hind II, which was more resistant to cleavage than the other Hind II sites in wild type mtDNA. It had two cleavage sites for Hha I and gave two Hha fragments, which were arranged alternatively. Digestion with Hae III gave four fragments and these fragments were mapped.Mitochondrial DNA of this mutant showed a loss of heterogeneity in a melting profile. It melted within a narrow range of temperature, which was similar to that of poly dA·poly dT. Its differential melting curve was significantly different from that of wild type mtDNA.Mapping of mtDNA of a wild type yeast was carried out with restriction endonucleases. Fragments of mtDNA, which were isolated from petites carrying oligomycin-erythromycin-chloramphenicol-resistance and erythromycin-chloramphenicol resistance were also mapped. Loci of oligomycin-resistance, erythromycin-resistance and chloramphenicol-resistance were investigated based on the maps of Eco R I fragments and Hind II fragments.     

Russian ethnic history inferred from mitochondrial DNA diversity

With the aim of gaining insight into the genetic history of the Russians, we have studied mitochondrial DNA diversity among a number of modern Russian populations. Polymorphisms in mtDNA markers (HVS-I and restriction sites of the coding region) of populations from 14 regions within present-day European Russia were investigated. Based on analysis of the mitochondrial gene pool geographic structure, we have identified three different elements in it and a vast "intermediate" zone between them. The analysis of the genetic distances from these elements to the European ethnic groups revealed the main causes of the Russian mitochondrial gene pool differentiation. The investigation of this pattern in historic perspective showed that the structure of the mitochondrial gene pool of the present-day Russians largely conforms to the tribal structure of the medieval Slavs who laid the foundation of modern Russians. Our results indicate that the formation of the genetic diversity currently observed among Russians can be traced to the second half of the first millennium A.D., the time of the colonization of the East European Plain by the Slavic tribes. Patterns of diversity are explained by both the impact of the native population of the East European Plain and by genetic differences among the early Slavs.     

A modified procedure for isolation of yeast mitochondrial DNA

A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed. It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA. The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven. The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances.     

An increase in mitochondrial DNA promotes nuclear DNA replication in yeast

Coordination between cellular metabolism and DNA replication determines when cells initiate division. It has been assumed that metabolism only plays a permissive role in cell division. While blocking metabolism arrests cell division, it is not known whether an up-regulation of metabolic reactions accelerates cell cycle transitions. Here, we show that increasing the amount of mitochondrial DNA accelerates overall cell proliferation and promotes nuclear DNA replication, in a nutrient-dependent manner. The Sir2p NAD⁺-dependent de-acetylase antagonizes this mitochondrial role. We found that cells with increased mitochondrial DNA have reduced Sir2p levels bound at origins of DNA replication in the nucleus, accompanied with increased levels of K9, K14-acetylated histone H3 at those origins. Our results demonstrate an active role of mitochondrial processes in the control of cell division. They also suggest that cellular metabolism may impact on chromatin modifications to regulate the activity of origins of DNA replication.