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.     

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.     

Abnormal Mitochondrial Genomes in Yeast Restored to Respiratory Competence

When crosses are performed between newly arisen, spontaneous petite mutants of Saccharomyces cerevisiae, respiratory competent (restored) colonies can form. Some of the restored colonies are highly sectored and produce large numbers of petite mutants. The high-frequency petite formation trait is inherited in a non-Mendelian manner, and elimination of mitochondrial DNA from these strains results in the loss of the trait. These results indicate that abnormal mitochondrial genomes are sometimes formed during restoration of respiratory competence. It is hypothesized that these abnormalities result either from recombination between mitochondrial DNA fragments to produce molecules having partial duplications contained on inverted or transposed sequences, or else recombinational "hot spots" have been expanded.     

Fine structure of the 21S ribosomal RNA region on yeast mitochondrial DNA. II. The organization of sequences in petite mitochondrial DNAs carrying genetic markers from the 21S region.

We have investigated the organization of sequences in ten rho- petite mtDNAs by restriction enzyme analysis and electron microscopy. From the comparison of the physical maps of the petite mtDNAs with the physical map of the mtDNA of the parental rho+ strain we conclude that there are at least three different classes of petite mtDNAs: I. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA. II. Head-to-tail repeats of an (almost) continuous segment of the rho+ mtDNA with a terminal inverted duplication. III. Mixed repeats of an (almost) continuous rho+ mtDNA segment. In out petite mtDNAs of the second type, the inverted duplications do not cover the entire conserved rho+ mtDNA segment. We have found that the petite mtDNAs of the third type contain a local inverted duplication at the site where repeating units can insert in two orientations. At least in one case this local inverted duplication must have arisen by mutation. The rearrangements that we have found in the petite mtDNAs do not cluster at specific sites on the rho+ mtDNA map. Large rearrangements or deletions within the conserved rho+ mtDNA segment seem to contribute to the suppressiveness of a petite strain. There is also a positive correlation between the retention of certain segments of the rho+ mtDNA and the suppressiveness of a petite strain. We found no correlation between the suppressiveness of a petite strain and its genetic complexity. The relevance of these findings for the mechanism of petite induction and the usefulness of petite strains for the physical mapping of mitochondrial genetic markers and for DNA sequence analysis are discussed.     

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.     

The nucleotide sequence of the mitochondrial DNA genome of an abundant petite mutant of Saccharomyces cerevisiae carrying the ori1 replication origin

We have determined the 903 bp nucleotide sequence of the mitochondrial DNA genome of a Saccharomyces cerevisiae petite mutant BB5. This petite, containing the 265 nucleotide ori1 region, is representative of a class of petites arising at exceptionally high frequency within the population of spontaneous petites derived from a particular mit- strain Mb12. The DNA sequences of both the ori1 region and the flanking intergenic regions have been compared to those of the corresponding regions of mtDNA in a previously reported petite strain, a1/1R/1 of Bernardi's laboratory, that has a similar (880 bp) repeat unit. The BB5 petite genome carries a canonical ori1 sequence that is identical in both petite mtDNAs, but the flanking intergenic sequences show significant differences between the two petite strains. The divergence is considered to arise from differences in the sequences flanking ori1 in the respective parent strains.     

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit- mutant of Saccharomyces cerevisiae

In cultures of the mit- mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit- cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a 32P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the ori1 sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

Biogenesis of Mitochondria: Analysis of Deletion of Mitochondrial Antibiotic Resistance Markers in Petite Mutants of Saccharomyces cerevisiae

Yeast strains carrying markers in several mitochondrial antibiotic resistance loci have been employed in a study of the retention and deletion of mitochondrial genes in cytoplasmic petite mutants. An assessment is made of the results in terms of the probable arrangement and linkage of mitochondrial genetic markers. The results are indicative of the retention of continuous stretches of the mitochondrial genome in most petite mutants, and it is therefore possible to propose a gene order based on co-retention of different markers. The order par, mik1, oli1 is suggested from the petite studies in the case of three markers not previously assigned an unambiguous order by analysis of mitochondrial gene recombination. The frequency of separation of markers by deletion in petites was of an order similar to that obtained by recombination in polar crosses, except in the case of the ery1 and cap1 loci, which were rarely separated in petite mutants. The deletion or retention of the locus determining polarity of recombination (ω) was also demonstrated and shown to coincide with deletion or retention of the ery1, cap1 region of the mitochondrial genome. Petites retaining this region, when crossed with rho⁺ strains, display features of polarity of recombination and transmission similar to the parent rho⁺ strain. By contrast a petite determined to have lost the ω⁺ locus did not show normal polarity of marker transmission. Differences were observed in the relative frequency of retention of markers in a number of strains and also when comparing petites derived spontaneously with those obtained after ultraviolet light mutagenesis. By contrast, a similar pattern of marker retention was seen when comparing spontaneous with ethidium bromide-induced petites.     

Induction of petite yeast mutants by membrane-active agents.

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Mutator Activity of Petite Strains of SACCHAROMYCES CEREVISIAE

Petite strains in Saccharomyces exhibit enhanced spontaneous mutation rates of nuclear genes regardless of whether they are cytoplasmically or nuclearly inherited, or whether or not the cytoplasmic petite strains have mitochondrial DNA. In petite strains, the mutation rate for the nonsense allele lys1-1 is enhanced by a factor of 3-6 and for the missense allele his1-7 by a factor of 2 as compared with their grande counterparts. The reversion of a third allele, the putative frameshift mutation, hom3-10 , is not enhanced in a petite background. The results indicate that the spontaneous mutation rate of an organism can be altered by indirect intracellular influences.     

Inheritance and organisation of the mitochondrial genome differ between two Saccharomyces yeasts

Petite-positive Saccharomyces yeasts can be roughly divided into the sensu stricto, including Saccharomyces cerevisiae, and sensu lato group, including Saccharomyces castellii; the latter was recently studied for transmission and the organisation of its mitochondrial genome. S. castellii mitochondrial molecules (mtDNA) carrying point mutations, which confer antibiotic resistance, behaved in genetic crosses as the corresponding point mutants of S. cerevisiae. While S. castellii generated spontaneous petite mutants in a similar way as S. cerevisiae, the petites exhibited a different inheritance pattern. In crosses with the wild type strains a majority of S. castellii petites was neutral, and the suppressivity in suppressive petites was never over 50%. The two yeasts also differ in organisation of their mtDNA molecules. The 25,753 bp sequence of S. castellii mtDNA was determined and the coding potential of both yeasts is similar. However, the S. castellii intergenic sequences are much shorter and do not contain sequences homologous to the S. cerevisiae biologically active intergenic sequences, as ori/rep/tra, which are responsible for the hyper-suppressive petite phenotype found in S. cerevisiae. The structure of one suppressive S. castellii mutant, CA38, was also determined. Apparently, a short direct intergenic repeat was involved in the generation of this petite mtDNA molecule.     

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit− mutant of Saccharomyces cerevisiae

In cultures of the mit^? mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit^? cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a ³²P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the oril sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

A region of extreme instability in the mitochondrial genome of yeast.

About half of the spontaneous petite mutants produced by wild-type Saccharomyces cerevisiae strain B (as well as by several other strains) have the same defective mitochondrial genome. Its repeat unit is a segment, 2200 base pairs (bp) long, which derives from an excision between the origins of replication ori 2 and ori 7 of the wild-type genome, and contains a hybrid ori 2-ori 7 sequence. The spontaneous petites carrying this defective ori.h genome are supersuppressive , i.e., they very rapidly compete out the wild-type genome in crosses. The main reasons for the exceptional frequency of ori.h petites are an extremely high excision frequency, due to the extended homology between the two tandemly oriented ori sequences 265 bp long and the short distance separating them. Such an excision frequency is very strongly increased in petite genomes encompassing the ori 2-ori 7 region, because of their higher concentration in these ori sequences.     

Induction of petite yeast mutants by membrane-active agents

Ethanol proved to be a strong mutagenic agent of Saccharomyces mitochondrial DNA. Other active membrane solvents, such as tert-butanol, isopropanol, and sodium dodecyl sulfate, also turned out to be powerful petite mutation [rho-] inducers. Mutants defective in ergosterol synthesis (erg mutants) showed an extremely high frequency of spontaneous petite cells, suggesting that mitochondrial membrane alterations that were caused either by changes in its composition, as in the erg mutants, or by the effects of organic solvents resulted in an increase in the proportion of petite mutants. Wine yeast strains were generally more tolerant to the mutagenic effects of alcohols on mitochondrial DNA and more sensitive to the effect of sodium dodecyl sulfate than laboratory strains. However, resistance to petite mutation formation in laboratory strains was increased by mitochondrial transfer from alcohol-tolerant wine yeasts. Hence, the stability of the [rho+] mitochondrial DNA in either the presence or absence of solvents depends in part on the nature of the mitochondrial DNA itself. The low frequency of petite mutants found in wine yeast-laboratory yeast hybrids and the fact that the high frequency of petite mutants of a particular wine spore segregated meiotically indicated that many nuclear genes also play an important role in the mitochondrial genome in both the presence and absence of membrane solvents.     

Physical and genetic organization of Petite and Grande yeast mitochondrial DNA. III. High resolution melting and reassociation studies

Mitochondrial DNAs from 13 petite mutants have been analyzed by means of high-resolution melting and reassociation in solution, in an attempt to relate their physical chemical properties to the mitochondrial genotype, which displays various combinations of genetic marker deletions. The kinetic complexities of the petite mtDNAs were found to range from $\text{1}\text{3}$ down to $\text{1}\text{500}$ of that of the grande mtDNA; the loss in sequential complexity undergone by petite mtDNAs parallels the loss in mitochondrial genetic complexity. Melting profiles of petite mtDNAs can be resolved into well-defined peaks. Some of them are specific to the marker genes. The genotypic specificity increases as the sequential complexity of mtDNA decreases. The mtDNA region conferring resistance to erythromycin could in this way be shown to be characterized by two melting peaks, at 72 °C and 75 °C. The results are interpreted in terms of selective enrichment in gene-specific sequences.     

Mitochondrial DNA loss caused by ethanol in Saccharomyces flor yeasts.

Saccharomyces flor yeasts proliferate at the surface of sherry wine, which contains over 15% (vol) ethanol. Since ethanol is a powerful inducer of respiration-deficient mutants, this alcohol has been proposed to be the source of the high diversity found in the mitochondrial genomes of flor yeasts and other wine yeasts. Southern blot analysis suggests that mitochondrial DNA (mtDNA) polymorphic changes are due to minor lesions in the mitochondrial genome. As determined in this work by pulsed-field gel electrophoresis, restriction analysis, and Southern blot analysis, ethanol-induced petite mutants completely lack mtDNA (rho zero). Ethanol-induced changes in the mitochondrial genome that could explain the observed mtDNA polymorphism in flor yeasts were not found. The transfer of two different mtDNA variants from flor yeasts to a laboratory strain conferred in both cases an increase in ethanol tolerance in the recipient strain, suggesting that mtDNAs are probably subjected to positive selection pressure concerning their ability to confer ethanol tolerance.     

Surrogate origins of replication in the mitochondrial genomes of ori-zero petite mutants of yeast.

We have investigated the mitochondrial genome of eight ori-zero spontaneous petite mutants of Saccharomyces cerevisiae. The tandem repeat units of these genomes do not contain any of the seven canonical ori sequences of the wild-type genome. Instead, they contain one, or more, ori-S sequences. These 44-nucleotide long surrogate origins of replication are a subset of GC clusters characterized by a potential secondary fold with two sequences ATAG and GGAG , inserted in AT spacers, two AT base pairs just following them, a GC stem (broken in the middle, and, in most cases also near the base, by non-paired nucleotides), and a terminal loop. This structure is reminiscent of that of GC clusters A and B from canonical ori sequences and supports the view (Bernardi, 1982a ) that the GC clusters of the mitochondrial genome arose, by an expansion process, from the canonical ori sequences. Like the latter, ori-S sequences are present in both orientations, are located in intergenic regions, and can be used as excision sequences when tandemly oriented. Again as in the case of canonical ori sequences, the density of ori-S sequences on the repeat units of petite genomes are correlated with the replication efficiency of the latter, as assessed by the outcome of crosses with wild-type or petite tester strains.     

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.     

Mitochondrial genetics in perspective: the derivation of a genetic and physical map of the yeast mitochondrial genome.

The attainment of the map of functions coded in the yeast mitochondrial genome represents the end of an era of development in mitochondrial genetics. Following the earliest genetic studies, where first the respiration-deficient petite mutants, then subsequently the other types of mitochondrial mutants, were characterized, it was realized that a genetic approach to the questions of mitochondrial biogenesis and the genetic function of mtDNA would yield much useful information. A period of intensive investigation into the behavior of mitochondrial genes in genetic crosses followed, and it was concluded that the purely genetic techniques of transmissional and recombinational analysis could not yield a map of the genetic loci, although basic rules for mitochondrial genetic manipulation were established. The concurrent studies of the nature of the deletions in petite mtDNA led to the recognition that an analysis of the behavior of genetic loci in petite mutants would provide the method for genetically mapping the positions of loci in mtDNA where conventional genetic crosses between grande strains had failed. This thesis was first confirmed by our studies of the frequencies of coretention and loss of individual loci in large populations of petite isolates, which produced the first circular genetic map of drug resistance loci on mtDNA. Subsequent to this genetic mapping phase, we established a general procedure for determining the physical map position of any mitochondrial genetic locus or mtDNA sequence by introducing the use of a molecular library of petite mutants carrying physically and genetically defined segments of mtDNA. These petites can be tested for the retention or loss of genetic loci or particular nucleotide sequences. This general solution to the mapping problem and the physical map of the Saccharomyces cerevisiae mitochondrial genome obtained, which has been confirmed by studies using restriction enzymes, has provided the field with a molecular point of reference for the many current genetic and biochemical investigations into the structure and function of mtDNA in yeast.