The mitochondrial genomes of spontaneous orir petite mutants of yeast have rearranged repeat units organized as inverted tandem dimers

We have investigated the structure and organization of the mitochondrial genomes of two related orir (ori-rearranged) spontaneous petite mutants of Saccharomyces cerevisiae. In these mutant genomes every repeat unit contains an inverted terminal duplication harboring a second (inverted) ori sequence, and tandem pairs of repeat units alternate with tandem pairs in inverted orientation. We have shown that orir genomes are organized as the genomes with inverted repeat units of ethidium bromide (EtBr)-induced petites, and we have clarified the mechanism by which such mutant mitochondrial genomes arise.     

Temperature can reversibly modify the structure and the functional efficiency of ori sequences of the yeast mitochondrial genome

We have compared the suppressibility of three isonuclear spontaneous, cytoplasmic petite mutants of Saccharomyces cerevisiae, as measured at three temperatures, 23 degrees C, 28 degrees C and 33 degrees C. The three petites have mitochondrial genomes made up of repeat units which are about 400 bp in size, and carry an origin of replication, ori1. This ori sequence is intact in petite Z1, whereas it lacks GC cluster A in petite 26 and cluster A plus some contiguous nucleotides in petite 14. These deletions lead to the impossibility to form a stem-and-loop structure of the ori sequence, the 'A-B fold', which involves two GC clusters, A and B, and the nucleotides in between. Instead, a 'replacement fold', only involving AT base pairs, is feasible. In petites 14 and 26, suppressivity decreases when the temperature is raised from 28 degrees C to 33 degrees C, and increases when the temperature is lowered from 28 degrees C to 23 degrees C. In contrast, no changes are seen in petite Z1. These temperature effects correlate with the stability of the 'A-B fold' and the instability of the 'replacement folds'. Since suppressibility measures the replicative competitiveness of the petite genome relative to the wild-type genome, these results indicate that an environmental parameter, temperature, can reversibly affect the structure and the functional efficiency of ori sequences in vivo. The evolutionary implications of these findings are discussed.     

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.     

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.     

Regions flanking ori sequences affect the replication efficiency of the mitochondrial genome of ori+ petite mutants from yeast

The mitochondrial genomes of progenies from 26 crosses between 17 cytoplasmic, spontaneous, suppressive, ori+ petite mutants of Saccharomyces cerevisiae have been studied by electrophoresis of restriction fragments. Only parental genomes (or occasionally, genomes derived from them by secondary excisions) were found in the progenies of the almost 500 diploids investigated; no evidence for illegitimate, site-specific mitochondrial recombination was detected. One of the parental genomes was always found to be predominate over the other one, although to different extents in different crosses. This predominance appears to be due to a higher replication efficiency, which is correlated with a greater density of ori sequences on the mitochondrial genome (and with a shorter repeat unit size of the latter). Exceptions to the 'repeat-unit-size rule' were found, however, even when the parental mitochondrial genomes carried the same ori sequence. This indicates that noncoding, intergenic sequences outside ori sequences also play a role in modulating replication efficiency. Since in different petites such sequences differ in primary structure, size, and position relative to ori sequences, this modulation is likely to take place through an indirect effect on DNA and nucleoid structure.     

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.     

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.     

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.     

Biogenesis of mitochondria: XLI. Physical mapping of mitochondrial genetic markers in yeast

This paper describes the physical mapping of five antibiotic resistance markers on the mitochondrial genome of Saccharomyces cerevisiae. The physical separations between markers were derived from studies involving a series of stable spontaneous petite strains which were isolated and characterized for the loss or retention of combinations of the five resistance markers. DNA-DNA hybridization using ³²P-labelled grande mitochondrial DNA was employed to determine the fraction of grande mitochondrial DNA sequences retained by each of the defined petite strains.One petite clone retaining four of the markers in a segment comprising 36% of the grande genome was then chosen as a reference petite. The sequence homology between the mitochondrial DNA of this petite and that of the other petites was measured by DNA-DNA hybridization. For each petite, the total length of its genome derived by hybridization with grande mitochondrial DNA and the fraction of the grande genome retained in common with the reference petite, together with the genetic markers retained in common, were used to position the DNA segment of each petite relative to the reference petite genome. At the same time the relative physical location of the five markers on a circular genome was established. On the basis of the grande mitochondrial genome being defined as 100 units of DNA, the positions of the markers were determined to bo as follows, measuring from one end of the reference petite genome. chloramphenicol (cap1) ~ 0 units erythromycin (ery1) 0 to 15 units oligomycin (oli1) 18 to 19 units mikamycin (mik1) 22 to 25 units paromomycin (par1) 61 to 73 unitsThe general problems of mapping mitochondrial genetic markers by hybridizations involving petite mitochondrial DNA are discussed. Two very important features of petite genomes which could invalidate the interpretation of DNA-DNA hybridization experiments between petite mitochondrial DNAs are the possible presence in the reference petite of differentially amplified DNA sequences, and/or “new” sequences which are not present in the parent grande genome. A general procedure, which overcomes errors of interpretation arising from these two features is described.     

A genetic and biochemical analysis of petite mutations in yeast

A series of spontaneous cytoplasmic petite mutants was isolated from a grande strain of Saccharomyces cerevisiae doubly marked with the cytoplasmically inherited determinants to erythromycin and oligomycin resistance. The petites were characterized with regard to the genetic stability of these antibiotic resistance markers and to their degree of suppressivity. No relation was found between the genetic instability of a petite mutant and the degree of suppressivity exhibited by that mutant. Three petites of 19.4%, 57.4% and 90.4% suppressivity were selected and their mitochondrial DNA characterized with regard to molecular weight, buoyant density in analytical cesium chloride density gradients, and the percentage of the total cellular DNA represented by the mitochondrial DNA. From these results it appears that the molecular weight of the mitochondrial DNA of the petite strains examined is the same as that shown by the parental grande strain, regardless of the degree of suppressivity exhibited.     

Excision sequences in the mitochondrial genome of yeast

We report an analysis of the sequences used in the excision of the mitochondrial genomes of 22 spontaneous and ten ethidium bromide (EtBr)-induced Saccharomyces cerevisiae petite mutants. In all cases, excision sequences were found to be perfect direct repeats, often flanked on one or both sides by regions of patchy homology. Sequences used in the excision of the genomes of spontaneous petites were always located in the AT spacers and GC clusters of intergenic regions of the genome; the GC clusters corresponded to ori and ori^s sequences, namely to canonical and surrogate origins of DNA replication, respectively. In the case of the ethidium bromide-induced petites, excision sequences were found not only in intergenic sequences, but also in the introns and exons of mitochondrial genes.     

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.     

Preferential recombination between GC clusters in yeast mitochondrial DNA.

Yeast mitochondrial DNA molecules have long, AT-rich intergenic spacers punctuated by short GC clusters. GC-rich elements have previously been characterized by others as preferred sites for intramolecular recombination leading to the formation of subgenomic petite molecules. In the present study we show that GC clusters are favored sites for intermolecular recombination between a petite and the wild-type grande genome. The petite studied retains 6.5 kb of mitochondrial DNA reiterated tandemly to form molecules consisting of repeated units. Genetic selection for integration of tandem 6.5 kb repeats of the petite into the grande genome yielded a novel recombination event. One of two crossovers in a double exchange event occurred as expected in the 6.5 kb of matching sequence between the genomes, whereas the second exchange involved a 44 bp GC cluster in the petite and another 44 bp GC cluster in the grande genome 700 bp proximal to the region of homology. Creation of a mitochondrial DNA molecule with a repetitive region led to secondary recombination events that generated a family of molecules with zero to several petite units. The finding that 44 bp GC clusters are preferred as sites for intermolecular exchange adds to the data on petite excision implicating these elements as recombinational hotspots in the yeast mitochondrial genome.     

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 47

Summary This paper consolidates and refines the physical map of genetic loci previously established in our laboratory, by molecular analysis of seven genetically characterized new petites (deletion mutants of mtDNA). A modified DNA-DNA hybridization procedure employing filters simultaneously bound with mtDNA from two different petites has been used to measure the overlaps in mtDNA sequences between the different petite mutants.Thus, by analysis of three new petites carrying the antibiotic-resistance loci, ery1, cap1 and par1 on their mitochondrial genomes, it has now been possible to improve our estimation of the maximum distance between the cap1 and ery1 loci. The cap1, ery1 loci, and the 21S ribosomal RNA gene have now been mapped within 5 units in the same region (map position 0 to 5 units). Similarly, by analysis of four new petites carrying the O _II and/or par1 loci on their mtDNAs, the map position of the O _II locus is also more accurately determined within 2 units in a region (map position 34 to 36 units) between the par1 and ana1 loci. The positions of other loci including par1, the 15S ribosomal RNA gene, and some mit ^- loci are also discussed.We have thus extended our library of genetically and molecularly defined petite mutants, resulting in a set of petites having overlapping regions distributed throughout the entire wild-type mitochondrial genome, consistent with the idea that yeast mtDNA is physically circular.     

Evolutionarily selected replication origins: functional aspects and structural organization.

A selective replicative pressure occurs during the evolution of simian virus 40 variants. When the replication origin is duplicated as an inverted repeat, there is a dramatic enhancement of replication. Having regulatory sequences located between the inverted repeat of ori magnifies their enhancing effect on replication. A passage 20 variant and a passage 45 variant containing three pairs of an inverted repeat of ori replicated more efficiently than a passage 13 variant containing nine copies of ori arranged in tandem. A 69-base-pair cellular sequence inserted between inverted repeats of ori of both passage 40 and 45 variants enhanced simian virus 40 DNA replication. Differences in replication efficiencies became greater as the total number of replicating species was increased in the transfection mixture, under conditions where T antigen is limiting. In a competitive environment, sequences flanking the replication origin may be inhibitory to replication.