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     

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.     

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.     

Inversions in mitochondrial DNA of a petite mutant of yeast

Mitochondrial DNA of an erythromycin-resistant petite mutant of yeast, E734, showed physical maps of inversion, which occurred between two cross-over sites in the fragments Hae A and Hae B. The pair of cross-over sites was inferred to be accommodated within a repeat unit of E734 mtDNA during petite mutation by joining two fragments excised from non-adjacent region of wild type mtDNA.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

Mhr1p-dependent concatemeric mitochondrial DNA formation for generating yeast mitochondrial homoplasmic cells

Mitochondria carry many copies of mitochondrial DNA (mtDNA), but mt-alleles quickly segregate during mitotic growth through unknown mechanisms. Consequently, all mtDNA copies are often genetically homogeneous within each individual ("homoplasmic"). Our previous study suggested that tandem multimers ("concatemers") formed mainly by the Mhr1p (a yeast nuclear gene-encoded mtDNA-recombination protein)-dependent pathway are required for mtDNA partitioning into buds with concomitant monomerization. The transmission of a few randomly selected clones (as concatemers) of mtDNA into buds is a possible mechanism to establish homoplasmy. The current study provides evidence for this hypothesis as follows: the overexpression of MHR1 accelerates mt-allele-segregation in growing heteroplasmic zygotes, and mhr1-1 (recombination-deficient) causes its delay. The mt-allele-segregation rate correlates with the abundance of concatemers, which depends on Mhr1p. In G1-arrested cells, concatemeric mtDNA was labeled by [14C]thymidine at a much higher density than monomers, indicating concatemers as the immediate products of mtDNA replication, most likely in a rolling circle mode. After releasing the G1 arrest in the absence of [14C]thymidine, the monomers as the major species in growing buds of dividing cells bear a similar density of 14C as the concatemers in the mother cells, indicating that the concatemers in mother cells are the precursors of the monomers in buds.