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.     

DNA-binding proteins in yeast: Effect of growth phase and mitochondrial function

DNA-binding proteins of the yeast Saccharomyces cerevisiae have been examined by DNA-cellulose chromatography with the expectation that they should represent, in part, a subclass of those proteins which bind to or interact with the chromosomes in vivo. After a high speed supernatant of a deoxyribonuclease-treated cell lysate is passed through a column of calf thymus DNA-cellulose, the DNA-binding proteins are eluted with a discontinuous salt gradient. The DNA-binding proteins, which show a broad distribution in size when examined by electrophoresis on polyacrylamide slab-gels in the presence of sodium dodecyl sulfate, represent about 0.2–0.3% of the cell's protein corresponding to about 5 × 10⁹-molecular weight of protein per haploid cell. Our data demonstrate quantitative and qualitative changes in the spectrum of DNA-binding proteins which may be correlated with changes in growth rate, stage of the growth cycle and phenotypic (repressed versus derepressed) and genetic alterations in mitochondrial function (grandes versus petites). The largest change which we have noted in the spectrum of DNA-binding proteins is between glucose-grown log-phase grande cells and grande cells in stationary phase. In many of the comparisons made, a number of specific DNA-binding proteins are seen to vary by as much as 5–10-fold. From estimates of the number of molecules of a DNA-binding protein present in the cell, we conclude that the system we have described is capable of detecting less than 100 molecules per yeast cell; within the range of the level of the lac represser in Escherichia coli.     

The synthesis of yeast matrix mitochondrial enzymes is regulated by different levels of mitochondrial function

The role of mitochondria in the oxygen induction of a number of catabolic and mitochondrial enzymes (citrate synthase, NAD- and NADP-isocitrate dehydrogenases, oxoglutarate dehydrogenase, NAD-glutamate dehydrogenase) has been investigated in anaerobic yeast grown under different conditions. The patterns of variation of enzyme activity with oxygen and lipid content of the mitochondria and with antibiotics suggest that more than one control is operating. The inhibition produced by cycloheximide, which blocks protein translation, suggests that induction involves de novo protein synthesis, except for an initial 2-h induction of citrate synthase, which is insensitive to all antibiotics tested. Ethidium bromide prevents enzyme induction in lipid-depleted anaerobic yeast. Induction follows normal kinetics in lipid-supplemented cultures despite the ethidium bromide block in the development of respiratory ability. Enzyme induction is inhibited by chloramphenicol in both lipid-depleted and lipid-supplemented anaerobic yeast. On the basis of four results it can be postulated that the mitochondrial genome is involved in controlling the induction of enzymes synthesized on cytoplasmic ribosomes. This control might be exerted by a specific, mitochondrial product or might be the result of modulation by a secondary product of mitochondrial function.     

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.     

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.     

Search for a regulatory function of mitochondrial proteinases in yeast

Proteolytic activity in yeast mitochondrial membranes and soluble fractions was studied using a labeled substrate: [¹⁴C]dimethylcasein. Optimal activity for the two fractions was at pH 6 in phosphate buffer at 37°C. Activity was partially inhibited by adding 3 mmol/1 ATP. Activity was enhanced rapidly in both fractions when yeast cells were grown aerobically on a derepression medium and transfered on a repression medium in anaerobiosis. Mitochondrial proteinase mediation in the mitochondrial breackdown observed during transition from aerobical derepression to anaerobical repression is discussed.     

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.     

Variations in the amino acid composition of cyanophycin in the cyanobacterium Synechocystis sp. PCC 6308 as a function of growth conditions

Gas chromatography-mass spectrometry studies of the nitrogen isotopic composition of the N-trifluoroacetyl n-butyl ester derivatives of the amino acids from isolated hydrolyzed cyanophycin from ¹⁵N-enriched cells led to two major findings: (1) the amino acid composition of this granular polypeptide, isolated using procedures optimized for extracting and purifying cyanophycin from cells in the stationary growth phase, varied with the culture growth condition; (2) the rate of incorporation of exogenous nitrate differed for each nitrogen atom of the amino acid constituents of cyanophycin or cyanophycin-like polypeptide. Arginine and aspartic acid were the principle components of cyanophycin isolated from exponentially growing cells and from light-limited stationary phase cells, with glutamic acid as an additional minor component. The cyanophycin-like polypeptide from nitrogen-limited cells contained only aspartic and glutamic acids, but no arginine. The glutamic acid content decreased and arginine content increased as nitrate was provided to nitrogen-limited cells. These cells rapidly incorporated nitrate at different rates at each cyanophycin nitrogen site: guanidino nitrogens of arginine>aspartic acid >-amino nitrogen of arginine>glutamic acid. Little media-derived nitrogen was incorporated into cyanophycin of exponentially growing cells during one cellular doubling time.Abbreviations asp-TAB, glu-TAB, arg-TAB N-Trifluoroacetyl n-butyl ester derivatives of aspartic acid, glutamic acid and arginine, respectively - CAP chloramphenicol - CF correction factor - TFAA Trifluoroacetic anhydride - MBTFA N-Methyl-bis-trifluoroacetamide     

Physiological scenarios of programmed loss of mitochondrial DNA function and death of yeast

Recently it was convincingly shown that the yeast Saccharomyces cerevisiae does possess the basic modules of programmed cell death machinery. As programmed cell death is suicide for a unicellular organism, it is reasonable to assume that they trigger the program when the death is beneficial for the rest of the population. Not surprisingly, most of the scenarios of physiological death of S. cerevisiae, i.e. cell death in stationary culture, during meiosis, during mating, and driven by viruses are dependent on quorum sensing, meaning that they depend on the cell density. Here we also discuss possible mechanisms that govern fitness decline during replicative aging of S. cerevisiae cells. We argue that loss of mitochondrial DNA function that occurs during replicative aging is programmed and adaptive. Indeed, yeast cells with nonfunctional mitochondrial DNA are known to be extremely stress-resistant, and also the presence of a subpopulation of such cells might protect the culture from degeneration by preventing the fixation of opportunistic mutations.     

Flow dichroism of T7 DNA as a function of salt concentration

Measurements have been made on the flow dichroism of T7 DNA as a function of NaCl concentration. In high salt, our results are compatible with an optical factor of ?1.4 and a persistence length of 470 Å. The former is in agreement with expectations from the x-ray diffraction structure of fibrous B–DNA, and the latter is in the midrange of recent determinations. As salt concentration is decreased, the persistence length increases. The relation of our study to other recent investigations and with current theories of the electrostatic contribution to persistence length is discussed. We note that the separation of the electrostatic expansion factor into long- and short-range effects is somewhat arbitrary and might affect the interpretation of different experimental results in different ways. Finally, our hydrodynamic factors are consistent with a chain which is partially free-draining. This goes against the traditional interpretation but is in agreement with two recent observations.     

Sphingolipids and mitochondrial function in budding yeast

Background

Sphingolipids (SLs) are not only key components of cellular membranes, but also play an important role as signaling molecules in orchestrating both cell growth and apoptosis. In Saccharomyces cerevisiae, three complex SLs are present and hydrolysis of either of these species is catalyzed by the inositol phosphosphingolipid phospholipase C (Isc1p). Strikingly, mutants deficient in Isc1p display several hallmarks of mitochondrial dysfunction such as the inability to grow on a non-fermentative carbon course, increased oxidative stress and aberrant mitochondrial morphology.

Scope of review

In this review, we focus on the pivotal role of Isc1p in regulating mitochondrial function via SL metabolism, and on Sch9p as a central signal transducer. Sch9p is one of the main effectors of the target of rapamycin complex 1 (TORC1), which is regarded as a crucial signaling axis for the regulation of Isc1p-mediated events. Finally, we describe the retrograde response, a signaling event originating from mitochondria to the nucleus, which results in the induction of nuclear target genes. Intriguingly, the retrograde response also interacts with SL homeostasis.

Major conclusions

All of the above suggests a pivotal signaling role for SLs in maintaining correct mitochondrial function in budding yeast.

General significance

Studies with budding yeast provide insight on SL signaling events that affect mitochondrial function.     

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     

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.     

The curious history of yeast mitochondrial DNA

Forty years ago, soon after yeast mitochondrial DNA (mtDNA) was recognized, some animal versions of mtDNA were shown to comprise circular molecules. Supporting an idea that mitochondria had evolved from bacteria, this finding generated a dogmatic belief that yeast mtDNA was also circular, and the endless linear molecules actually observed in yeast were regarded as broken circles. This concept persisted for 30 years and has distorted our understanding of the true nature of the molecule.