Induction of petite mutations during germination and outgrowth of Saccharomyces cerevisiae ascospores.

The germination and outgrowth of Saccharomyces cerevisiae ascospores were studied by determining the sensitivity of the ascospores to the action of chemical mutagens. Survival of the ascospores after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment was low during the first 2 h of germination and then increased and remained constant. Survival of the ascospores after 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine-2HC1 (ICR-170) treatment was constant from 0 to 5 h, but as the ascospores completed outgrowth at 6 h they became more sensitive to killing by ICR-170. Survival of the ascospores remained high during treatment with 2-methoxy-6-chloro-9-(3-[ethyl-2-hydroxyethyl]aminopropylamino)acridine-2HC1 (ICR-170-OH) or 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide. The main classes of mutations screened for were petites and auxotrophs. The induction of petites and auxotrophs by MNNG was independent of the stage of germination and outgrowth treated. Petite induction by ICR-170 was dependent upon the stage of germination and outgrowth treated. The early hours of germination (0 to 3 h) were not sensitive to petite induction. However, there was maximal petite induction at 5 h into germination and outgrowth, followed by a decline. During this same time period, ICR-170 induced less than 1% auxotrophic colonies. This finding is very unusual because ICR-170 induced 15% auxotrophic colonies in starved log-phase cultures of S. cerevisiae. The acridine ICR-170-OH induced no mutations during germination and outgrowth of the ascospores. Ethidium bromide induced petites, and the petite frequency became maximal at 5 h of germination and outgrowth, a result similar to that obtained with ICR-170.     

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.     

Heterogeneity of mitochondrial DNA from Saccharomyces cerevisiae and genetic information for tRNA.

Mitochondrial DNA from wild-type Saccharomyces cerevisiae and from an "extreme" petite mutant were analyzed by hybridization of several tRNAs on DNA fragments of different buoyant density, obtained by sonication and fractionation on a CsCl gradient. The hybridization patterns show that the genes for tRNAser, tRNAphe, tRNAhis, tRNAval, tRNAileu are present on wild-type mitochondrial DNA, while only genes for tRNAser and tRNAhis are present on petite mitochondrial DNA; moreover the hybridization patterns indicate that these genes are not clustered and suggest that more than one gene might exist for tRNAser and tRNAhis.     

The formation of a defective small subunit of the mitochondrial ribosomes in petite mutants of Saccharomyces cerevisiae

The involvement of mitochondrial protein synthesis in the assembly of the mitochondrial ribosomes was investigated by studying the extent to which the assembly process can proceed in petite mutants of Saccharomyces cerevisiae which lack mitochondrial protein synthetic activity due to the deletion of some tRNA genes and/or one of the rRNA genes on the mtDNA. Petite strains which retain the 15-S rRNA gene can synthesize this rRNA species, but do not contain any detectable amounts of the small mitochondrial ribosomal subunit. Instead, a ribonucleoparticle with a sedimentation coefficient of 30 S (instead of 37 S) was observed. This ribonucleoparticle contained all the small ribosomal subunit proteins with the exception of the var1 and three to five other proteins, which indicates that the 30-S ribonucleoparticle is related to the small mitochondrial ribosomal subunit (37 S). Reconstitution experiments using the 30-S particle and the large mitochondrial ribosomal subunit from a wild-type yeast strain indicate that the 30-S particle is not active in translating the artificial message poly(U). The large mitochondrial ribosomal subunit was present in petite strains retaining the 21-S rRNA gene. The petite 54-S subunit is biologically active in the translation of poly(U) when reconstituted with the small subunit (37 S) from a wild-type strain. The above results indicate that mitochondrial protein synthetic activity is essential for the assembly of the mature small ribosomal subunit, but not for the large subunit. Since the var1 protein is the only mitochondrial translation product known to date to be associated with the mitochondrial ribosomes, the results suggest that this protein is essential for the assembly of the mature small subunit.     

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.     

Ribonucleic acid polymerase of germinating Bacillus cereus T.

It appears that a de novo synthesis of the deoxyribonucleic acid-dependent ribonucleic acid-polymerase in Bacillus cereus T takes place fairly late in outgrowth, at the onset of the vegetative cycle. Therefore, the ribonucleic acid-polymerase used by germinating spores is the one carried on from sporulating cells. However, the sporal enzyme is less soluble that the vegetative one, and its "core" is bound to two extra peptides. This complexing to other molecules could play a role in the regulation of gene expression during germination.     

Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein.

Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins.     

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe

Spores from Schizosaccharomyces pombe contain neutral and acid trehalases. When spores from strains disrupted for ntp1(+), which encodes neutral trehalase, were induced to germinate, the onset of the process was markedly delayed as compared to wild-type spores. Further outgrowth was also reduced. Dormant spores lacking neutral trehalase contained twice the amount of trehalose present in wild-type spores and mobilised the intracellular pool of trehalose at a slower rate during germination. Inhibition by phloridzin of the sporulation-specific acid trehalase in ntp1-disrupted spores arrested germination completely while prompting no effect on wild-type spores. These results suggest that the two trehalase enzymes may support the utilisation of trehalose during germination but neutral trehalase is required for a more rapid and efficient process.     

Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation.

Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.     

Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation

Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance. The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures. Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway). Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca. 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance. Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents. All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.     

Life Cycle of Neurospora crassa Viewed by Scanning Electron Microscopy

Scanning electron microscopy was used to examine the major stages of the life cycle of two wild-type strains of Neurospora crassa Shear and Dodge (St. Lawrence 3.1a and 74A): mycelia, protoperithecium formation, perithecia, ascospores, ascospore germination and outgrowth, macro and microconidia, and germination and outgrowth of macroconidia. Structures seen at the limit of resolution of bright-field and phase-contrast microscopes, e.g., the ribbed surface of ascospores, are well resolved. New details of conidial development and surface structure are revealed. There appears to be only one distinguishable morphological difference between the two strains. The pattern of germination and outgrowth which seems relatively constant for strain 74A or strain 3.1a, appears to be different for each. Conidia from strain 3.1a almost always germinate from a site between interconidial attachment points; whereas the germ tubes of strain 74A usually emerge from or very near the interconidial attachment site. These germination patterns usually do not segregate 2:2 in asci dissected in order. This observation suggests that conidial germination pattern is not under the control of a single gene.     

Mitochondrial DNA replication in petite mutants of yeast: Resistance to inhibition by ethidium bromide, berenil and euflavine

Summary Mitochondrial DNA (mtDNA) replication in petite mutants ofSaccharomyces cerevisiae is generally less sensitive to inhibition by ethidium bromide than in grande (respiratory competent) cells. In every petite that we have examined, which retain a range of different grande mtDNA sequences, this general phenomenon has been demonstrated by measurements of the loss of mtDNA from cultures grown in the presence of the drug. The resistance is also demonstrable by direct analysis of drug inhibition of mtDNA replication in isolated mitochondria. Furthermore, the resistance to ethidium bromide is accompanied, in every case tested, by cross-resistance to berenil and euflavine, although variations in the levels of resistance are observed.In one petite the level of in vivo resistance to the three drugs was very similar (4-fold over the grande parent) whilst another petite was mildly resistant to ethidium bromide and berenil (each 1.6-fold over the parent) and strongly resistant (nearly 8-fold) to inhibition of mtDNA replication by euflavine. The level of resistance to ethidium bromide in several other petite clones tested was found to vary markedly. Using genetic techniques it is possible to identify those petites which display an enhanced resistance to ethidium bromide inhibition of mtDNA replication.It is considered that the general resistance of petites arises because a product of mitochondrial protein synthesis is normally involved in facilitating the inhibitory action of these drugs on mtDNA synthesis in grande cells. The various levels of resistance in petites may be modulated by the particular mtDNA sequences retained in each petite.     

Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae

Ethidium bromide is known to be a powerful mutagen for the induction of cytoplasmically inherited petite mutations in yeast. The effect of ethidium bromide on the degree of suppressiveness of the induced mutants as a function of exposure time is described. The mitochondrial DNA of 20 ethidium bromide-induced petite mutants has been studied to determine its absence or presence and its buoyant density. Ten mutants, in which we were not able to detect any mitochondrial DNA, were neutral petites. The 10 remaining mutants with mitochondrial DNA simultaneously showed a measurable degree of suppressiveness. It was not possible to correlate the buoyant density of the mutant mitochondrial DNA with the degree of suppressiveness.This study was supported in part by USPHS grant GM 10017. G.M. received a Fulbright Travel Grant.     

Lethal mitochondrial genotypes in Podospora anserina: A model for senescence

Summary Crosses between spg1 and spg2, two mitochondrial mutants of Podospora anserina, yield a new type of strain, called pseudo wild-type (PSW), in addition to wild-type recombinants. PSW strains are characterized by a variable phenotype for germination of ascospores and a variable longevity. By autofecondation, PSW strains yield early lethal strains (which die soon after the germination of the spores and so cannot be used for further studies), short-lived strains (which stop their vegetative growth after several centimeters) and long-lived strains (which grow longer than 16 cm). Genetic analysis of the last two categories shows that the PSW phenotype corresponds to a new mitochondrial genotype resulting from the interaction of the two parental mitochondrial genomes.Variability in the longevity of PSW strains is interpretated as the result of a high rate of mutation of their mitochondrial genome into a lethal and suppressive genome, similar to that of the mitochondrial rho ^- suppressive mutant of yeast. Furthermore, on the basis of the striking similarities observed between short-lived PSW strains and senescent cultures of Podospora anserina, we propose that commitment and development of senescence in wild-type strains of Podospora anserina would result, in a similar way, of spontaneous suppressive rho ^--like mitochondrial mutations.     

The Bacillus anthracis SleL (YaaH) protein is an N-acetylglucosaminidase involved in spore cortex depolymerization

Bacillus anthracis spores, the infectious agents of anthrax, are notoriously difficult to remove from contaminated areas because they are resistant to many eradication methods. These resistance properties are due to the spore's dehydration and dormancy and to the multiple protective layers surrounding the spore core, one of which is the cortex. In order for B. anthracis spores to germinate and resume growth, the cortex peptidoglycan must be depolymerized. This study reports on analyses of sleL (yaaH), which encodes a cortex-lytic enzyme. The inactivation of sleL does not affect vegetative growth, spore viability, or the initial stages of germination, including dipicolinic acid release. However, mutant spores exhibit a slight delay in the loss of optical density compared to that of wild-type spores. Mutants also retain more diaminopimelic acid and N-acetylmuramic acid during germination than wild-type spores, suggesting that the cortex peptidoglycan is not being hydrolyzed as rapidly. This finding is supported by high-pressure liquid chromatography analysis of the peptidoglycan structure used to confirm that SleL acts as an N-acetylglucosaminidase. When sleL is inactivated, the cortex peptidoglycan is not depolymerized into small muropeptides but instead is retained within the spore as large fragments. In the absence of the sleL-encoded N-acetylglucosaminidase, other cortex-lytic enzymes break down the cortex peptidoglycan sufficiently to allow rapid germination and outgrowth.     

Elevated Levels of Petite Formation in Strains of SACCHAROMYCES CEREVISIAE Restored to Respiratory Competence. II. Organization of Mitochondrial Genomes in Strains Having High and Moderate Frequencies of Petite Mutant Formation

Restriction enzyme analysis of aberrant mtDNA molecules in restored strains of Saccharomyces cerevisiae that displays an elevated level of petite formation has shown the occurrence of novel junction fragments and nonstoichiometric amounts for some unaltered bands. Five aberrant mitochondrial genomes from high-frequency petite-forming (hfp) strains (greater than 60% petites per generation) contain like-oriented duplications and single copy regions. High-frequency petite formation is postulated to arise from increased intramolecular recombination between duplicated segments. Mitochondrial DNA structures in two other hfp strains cannot be easily interpreted and might arise from intramolecular recombination. Mitochondria DNA from moderate-frequency petite-forming (mfp) strains (5-16% petites per generation) contains inverted duplications in two cases. The elevated petite formation is postulated to arise from homologous recombination between directly repeated sequences. In mtDNA from one mfp strain, deletion end-points have been shown to overlap. Such deletion endpoint overlap is postulated to be required for the maintenance of the tandem duplication in hfp strains. Two regions of the wild-type mtDNA (between cyb and oli2 and between SrRNA and oxi2) appear to be dispensable for mitochondrial function.     

Incompatibility of linear DNA killer plasmids pGKL1 and pGKL2 from Kluyveromyces lactis with mitochondrial DNA from Saccharomyces cerevisiae.

Two linear killer plasmids (pGKL1 and pGKL2) from Kluyveromyces lactis stably replicated and expressed the killer phenotype in a neutral petite mutant [( rho0]) of Saccharomyces cerevisiae. However, when cytoplasmic components were introduced by cytoduction from a wild-type [( rho+]) strain of S. cerevisiae, the linear plasmids became unstable and were frequently lost from the cytoductant cells during mitosis, giving rise to nonkiller clones. The phenomenon was ascribed to the incompatibility with the introduced S. cerevisiae mitochondrial DNA (mtDNA), because the plasmid stability was restored by [rho0] mutations in the cytoductant cells. Incompatibility with mtDNA was also apparent for the transmission of plasmids into diploid progeny in crosses between killer cells carrying the pGKL plasmids and [rho+] nonkiller cells lacking the plasmids. High-frequency transmission of the plasmids was observed in crosses lacking mtDNA [( rho0] by [rho0] crosses) and in crosses involving mutated mtDNA with large deletions of various regions of mitochondrial genome. In contrast, mutated mtDNA from various mit- mutations also exerted the incompatibility effect on the transmission of plasmids. Double-stranded RNA killer plasmids were stably maintained and transmitted in the presence of wild-type mtDNA and stably coexisted with pGKL killer plasmids in [rho0] cells of S. cerevisiae.     

The transmission disadvantage of yeast mitochondrial intergenic mutants is eliminated in the mgt1 (cce1) background.

A trans-acting element, MGT1 (also called CCE1), has previously been shown to be required in Saccharomyces cerevisiae for the preferential transmission of petite mitochondrial DNA (mtDNA) molecules over wild-type mtDNA molecules. In the present study a possible role of this nuclear gene in the transmission of mtDNA from various respiration-competent mutants was studied. Several of these mutants, lacking one or the other of two biologically active mitochondrial intergenic sequences, were employed in genetic crosses. When these deletion mutants were crossed to the parental wild-type strain in the MGT1/CCE1 background, the progeny contained predominantly wild-type mtDNA molecules. When crosses were performed in the mgt1/cce1 background, the parental molecules interacted in zygotes and underwent homologous recombination but wild-type and intergenic-deletion alleles were transmitted with equal frequencies.     

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae.

Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.