Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

Rapid,selective digestion of mitochondrial DNA in accordance with the matA hierarchy of multiallelic mating types in the mitochondrial inheritance of Physarum polycephalum

Although mitochondria are inherited uniparentally in nearly all eukaryotes, the mechanism for this is unclear. When zygotes of the isogamous protist Physarum polycephalum were stained with DAPI, the fluorescence of mtDNA in half of the mitochondria decreased simultaneously to give small spots and then disappeared completely approximately 1.5 hr after nuclear fusion, while the other mitochondrial nucleoids and all of the mitochondrial sheaths remained unchanged. PCR analysis of single zygote cells confirmed that the loss was limited to mtDNA from one parent. The vacant mitochondrial sheaths were gradually eliminated by 60 hr after mating. Using six mating types, the transmission patterns of mtDNA were examined in all possible crosses. In 39 of 60 crosses, strict uniparental inheritance was confirmed in accordance with a hierarchy of relative sexuality. In the other crosses, however, mtDNA from both parents was transmitted to plasmodia. The ratio of parental mtDNA was estimated to be from 1:1 to 1:10(-4). Nevertheless, the matA hierarchy was followed. In these crosses, the mtDNA was incompletely digested, and mtDNA replicated during subsequent plasmodial development. We conclude that the rapid, selective digestion of mtDNA promotes the uniparental inheritance of mitochondria; when this fails, biparental inheritance occurs.     

A Nuclear Mutation Reversing a Biased Transmission of Yeast Mitochondrial DNA

The highly biased transmission of ρ(-) mitochondrial DNA that occurs in hypersuppressive matings between ρ(-) and ρ(+) cells of the yeast Saccharomyces cerevisiae is thought to be a consequence of the replication advantage of the ρ(-) mtDNA. A nuclear gene, MGT1, that is required for this displacement of ρ(+) mtDNA from zygotic clones has been identified through mutation. When one haploid parent carries the mgt1 allele, transmission of ρ(-) mtDNA is substantially reduced. When both haploid parents carry the mgt1 allele, ρ(-) mtDNA is essentially eliminated from the zygotic progeny. Thus in the absence of the MGT1 gene there is a switch in the transmission bias; ρ(+) mtDNA rather than the hypersuppressive ρ(-) mtDNA is inherited by most zygotic clones. In contrast to its semi-dominant behavior in haploid matings, mgt1 behaves as a recessive allele in diploid matings since the ρ(+) genome in MGT1/mgt1 diploids is efficiently displaced when mated with a MGT1/mgt1 hypersuppressive ρ(-) diploid strain. We find that ρ(+) genomes can be comaintained along with hypersuppressive ρ(-) mtDNA for extended periods in clonal lines derived from MGT1 X mgt1 matings. However, as expected from the recessive nature of the mgt1 mutation, these ρ(+) genomes are eventually eliminated. Our work indicates that MGT1 plays a crucial role in the competition for inheritance between hypersuppressive ρ(-) mtDNAs and the ρ(+) mitochondrial genome. The MGT1 gene product may be a component of a mtDNA replication system that acts preferentially at the rep sequences found in hypersuppressive mtDNAs.     

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.     

Use of lycorine and DAPI staining in Saccharomyces cerevisiae to differentiate between rho0 and rho- cells in a cce1/delta cce1 nuclear background

In the yeast Saccharomyces cerevisiae, mutants are viable with large deletions (rho-), or even complete loss of the mitochondrial genome (rho0). One class of rho- mutants, which is called hypersuppressive, is characterised by a high transmission of the mutated mitochondrial genome to the diploid progeny when mated to a wild-type (rho+) haploid. The nuclear gene CCE1 encodes a cruciform cutting endonuclease, which is located in the mitochondrion and is responsible for the highly biased transmission of the hypersuppressive rho- genome. CCE1 is a Holliday junction specific endonuclease that resolves recombination intermediates in mitochondrial DNA. The cleavage activity shows a strong preference for cutting after a 5'-CT dinucleotide. In the absence of the CCE1 gene product, the mitochondrial genomes remain interconnected and have difficulty segregating to the daughter cells. As a consequence, there is an increase in the fraction of daughter cells that are rho0. In this paper we demonstrate the usefulness of lycorine, together with staining by 4',6-diamidino-2-phenylindole (DAPI), to assay for the mitotic stability of a variety of mitochondrial genomes. We have found that rho+ and rho- strains that contain CT sequences produce a large fraction of rho0 progeny in the absence of CCE1 activity. Only those rho- mitochondrial genomes lacking the CT recognition sequence are unaffected by the cce1 allele.     

Elevated Levels of Petite Formation in Strains of SACCHAROMYCES CEREVISIAE Restored to Respiratory Competence. I. Association of Both High and Moderate Frequencies of Petite Mutant Formation with the Presence of Aberrant Mitochondrial DNA

When recently arisen spontaneous petite mutants of Saccharomyces cerevisiae are crossed, respiratory competent diploids can be recovered. Such restored strains can be divided into two groups having sectored or unsectored colony morphology, the former being due to an elevated level of spontaneous petite mutation. On the basis of petite frequency, the sectored strains can be subdivided into those with a moderate frequency (5–16%) and those with a high frequency (>60%) of petite formation. Each of the three categories of restored strains can be found on crossing two petites, suggesting either that the parental mutants contain a heterogeneous population of deleted mtDNAs at the time of mating or that different interactions can occur between the defective molecules. Restriction endonuclease analysis of mtDNA from restored strains that have a wild-type petite frequency showed that they had recovered a wild-type mtDNA fragmentation pattern. Conversely, all examined cultures from both categories of sectored strains contained aberrant mitochondrial genomes that were perpetuated without change over at least 200 generations. In addition, sectored colony siblings can have different aberrant mtDNAs. The finding that two sectored, restored strains from different crosses have identical but aberrant mtDNAs provides evidence for preferred deletion sites from the mitochondrial genome. Although it appears that mtDNAs from sectored strains invariably contain duplications, there is no apparent correlation between the size of the duplication and spontaneous petite frequency.     

Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

Influence of a mitochondrial genetic defect on capacitative calcium entry and mitochondrial organization in the osteosarcoma cells

Effects of T8993G mutation in mitochondrial DNA (mtDNA), associated with neurogenical muscle weakness, ataxia and retinitis pigmentosa (NARP), on the cytoskeleton, mitochondrial network and calcium homeostasis in human osteosarcoma cells were investigated. In 98% NARP and rho(0) (lacking mtDNA) cells, the organization of the mitochondrial network and actin cytoskeleton was disturbed. Capacitative calcium entry (CCE) was practically independent of mitochondrial energy status in osteosarcoma cell lines. The significantly slower Ca(2+) influx rates observed in 98% NARP and rho(0), in comparison to parental cells, indicates that proper actin cytoskeletal organization is important for CCE in these cells.     

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.     

Patterns of mitochondrial sorting in yeast zygotes.

Inheritance of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae is usually biparental. Pedigree studies of zygotic first buds indicate limited mixing of wild-type (p+) parental mtDNAs: end buds are frequently homoplasmic for one parental mtDNA, while heteroplasmic and recombinant progeny usually arise from medial buds. In crosses involving certain petites, however, mitochondrial inheritance can be uniparental. In this study we show that mitochondrial sorting can be influenced by the parental mtDNAs and have identified intermediates in the process. In crosses where mtDNA mixing is limited and one parent is prelabeled with the matrix enzyme citrate synthase 1 (CS1), the protein freely equilibrates throughout the zygote before the first bud has matured. Furthermore, if one parent is p0 (lacking mtDNA), mtDNA from the p+ parent can also equilibrate; intracellular movement of mtDNA is unhindered in this case. Surprisingly, in zygotes from a p0 CS1+ x p+ CS1- cross, CS1 is quantitatively translocated to the p+ end of the zygote before mtDNA movement; subsequently, both components equilibrate throughout the cell. This initial vectorial transfer does not require respiratory function in the p+ parent, although it does not occur if that parent is p-. Mouse dihydrofolate reductase (DHFR) present in the mitochondrial matrix can also be vectorially translocated, indicating that the process is general. Our data suggest that in zygotes mtDNA movement may be separately controlled from the movement of bulk matrix constituents.     

Biparental mitochondrial DNA inheritance in the parasitic trematode Schistosoma mansoni

The maternal inheritance of mitochondrial DNA (mtDNA) in eukaryotic organisms occurs because of the selective destruction of paternal mtDNA molecules that may be present in the zygote. The elimination of sperm mtDNA is less efficient in interspecific crosses, and biparental inheritance of mtDNA has been observed in a variety of species. Because interspecific crosses are likely to be extremely rare in nature, parental inheritance of mtDNA has been deemed of little relevance to population genetics. The mtDNA of the parasitic trematode Schistosoma mansoni was examined for its utility in addressing epidemiological questions related to the transmission and spread of schistosomiasis. Prior to embarking on such experiments, we sought to confirm the mode of inheritance of this molecule using the highly polymorphic mtDNA minisatellite as a marker. In 3 separate crosses, mtDNA apparently identical to paternal DNA was observed in some individuals of the F2 and F3 generations. These observations thus suggest the intraspecific paternal inheritance of mtDNA across multiple generations in Schistosoma mansoni.     

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.     

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis

Summary The inheritance of both the mitochondrial DNA (mtDNA) and the nuclear-encoded extrachromosomal ribosomal DNA (rDNA) has been studied in the myxomycete, Didymium iridis, by DNA-DNA hybridization of labeled probes to total DNA at various stage of the life cycle. Both the mtDNA and rDNA populations rapidly become homogeneous in individuals, but there is a qualitative difference in the patterns of inheritance of these two molecules. One parental rDNA type was preferentially inherited in all crosses; selective replication of this molecule is tentatively proposed as the mechanism of inheritance. In contrast, either parental mtDNA type could be inherited. Since the inherited population of parental mtDNA molecules are not partitioned into cells in this coenocytic organism, no known mechanism of inheritance can explain the rapid and apparently random loss of one parental mtDNA type in individuals.     

Uncoupling the roles of the SUV3 helicase in maintenance of mitochondrial genome stability and RNA degradation

Yeast SUV3 is a nuclear encoded mitochondrial RNA helicase that complexes with an exoribonuclease, DSS1, to function as an RNA degradosome. Inactivation of SUV3 leads to mitochondrial dysfunctions, such as respiratory deficiency; accumulation of aberrant RNA species, including excised group I introns; and loss of mitochondrial DNA (mtDNA). Although intron toxicity has long been speculated to be the major reason for the observed phenotypes, direct evidence to support or refute this theory is lacking. Moreover, it remains unknown whether SUV3 plays a direct role in mtDNA maintenance independently of its degradosome activity. In this paper, we address these questions by employing an inducible knockdown system in Saccharomyces cerevisiae with either normal or intronless mtDNA background. Expressing mutants defective in ATPase (K245A) or RNA binding activities (V272L or ΔCC, which carries an 8-amino acid deletion at the C-terminal conserved region) resulted in not only respiratory deficiencies but also loss of mtDNA under normal mtDNA background. Surprisingly, V272L, but not other mutants, can rescue the said deficiencies under intronless background. These results provide genetic evidence supporting the notion that the functional requirements of SUV3 for degradosome activity and maintenance of mtDNA stability are separable. Furthermore, V272L mutants and wild-type SUV3 associated with an active mtDNA replication origin and facilitated mtDNA replication, whereas K245A and ΔCC failed to support mtDNA replication. These results indicate a direct role of SUV3 in maintaining mitochondrial genome stability that is independent of intron turnover but requires the intact ATPase activity and the CC conserved region.     

The plant-specific ssDNA binding protein OSB1 is involved in the stoichiometric transmission of mitochondrial DNA in Arabidopsis

Plant mitochondrial genomes exist in a natural state of heteroplasmy, in which substoichiometric levels of alternative mitochondrial DNA (mtDNA) molecules coexist with the main genome. These subgenomes either replicate autonomously or are created by infrequent recombination events. We found that Arabidopsis thaliana OSB1 (for Organellar Single-stranded DNA Binding protein1) is required for correct stoichiometric mtDNA transmission. OSB1 is part of a family of plant-specific DNA binding proteins that are characterized by a novel motif that is required for single-stranded DNA binding. The OSB1 protein is targeted to mitochondria, and promoter-beta-glucuronidase fusion showed that the gene is expressed in budding lateral roots, mature pollen, and the embryo sac of unfertilized ovules. OSB1 T-DNA insertion mutants accumulate mtDNA homologous recombination products and develop phenotypes of leaf variegation and distortion. The mtDNA rearrangements occur in two steps: first, homozygous mutants accumulate subgenomic levels of homologous recombination products; second, in subsequent generations, one of the recombination products becomes predominant. After the second step, the process is no longer reversible by backcrossing. Thus, OSB1 participates in controlling the stoichiometry of alternative mtDNA forms generated by recombination. This regulation could take place in gametophytic tissues to ensure the transmission of a functional mitochondrial genome.     

The mitochondrial genome of Chlamydomonas. II. Genetic analysis of non-mendelian obligate photautotrophic mutants

Summary Among a collection of obligate photoautotrophic (dark-dier,dk) mutants isolated inChlamydomonas reinhardtii, two have been found which are inherited in crosses to wild type in a non-Mendelian, biparental and apparently random fashion. F₁ progeny include not only cells which show thedk and wildtype parental phenotypes but also many which possess intermediate phenotypes between wild type anddk. When F₁ progeny withdk, intermediate or wild-type phenotype were backcrossed to wild type, thedk phenotype continued to be inherited in a biparental and random fashion. Upon selection, neither mutant formed stable clones producing onlydk progeny, suggesting that the two mutants segregatedk and wild-type progeny somatically and that the homozygousdk condition may be lethal. The biparental transmission of these two non-Mendeliandk mutations resembles the transmission of acriflavin-inducedminute mutations ofChlamydomonas and is distinct from the uniparentally inherited chloroplast mutations of this alga. Both thedk andminute mutations may alter mitochondrial DNA and thereby alter mitochondrial functions.     

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.     

Arabidopsis mitochondrial single-stranded DNA-binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination

Faithful DNA replication is one of the most essential processes in almost all living organisms. However, the proteins responsible for organellar DNA replication are still largely unknown in plants. Here, we show that the two mitochondrion-targeted single-stranded DNA-binding (SSB) proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA (mtDNA) maintenance, as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation. The mtDNA levels in mutants lacking SSB1 and/or SSB2 function were two- to four-fold higher than in the wild-type (WT), revealing a negative role for SSB1/2 in regulating mtDNA replication. Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA (POLIA) or POLIB in mtDNA replication, as mutation in either gene restored the high mtDNA copy number of the ssb1-1 mutant back to WT levels. In addition, SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mtDNA homologous recombination (HR). Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNA-BINDING PROTEIN1 (OSB1) during mtDNA replication, while SSB1 may act downstream of OSB1 and MUTS HOMOLOG1 for mtDNA HR. Overall, our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mtDNA stability via affecting DNA replication and DNA HR.     

Overlapping contributions of Msh1p and putative recombination proteins Cce1p, Din7p, and Mhr1p in large-scale recombination and genome sorting events in the mitochondrial genome of Saccharomyces cerevisiae

The mechanisms that govern mutation avoidance in the mitochondrial genome, though believed to be numerous, are poorly understood. The identification of individual genes has implicated mismatch repair and several recombination pathways in maintaining the fidelity and structural stability of mitochondrial DNA. However, the majority of genes in these pathways have not been identified and the interactions between different pathways have not been extensively studied. Additionally, the multicopy presence of the mitochondrial genome affects the occurrence and persistence of mutant phenotypes, making mitochondrial DNA transmission and sorting important factors affecting mutation accumulation. We present new evidence that the putative recombination genes CCE1, DIN7, and MHR1 have overlapping function with the mismatch repair homolog MSH1 in point mutation avoidance and suppression of aberrant recombination events. In addition, we demonstrate a novel role for Msh1p in mtDNA transmission, a role not predicted by studies of its nuclear homologs.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^? mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.