Mitochondrial DNA inheritance in sexual crosses of Pleurotus ostreatus

The inheritance of mitochondrial DNA (mtDNA) in sexual crosses was investigated to expand our understanding of the large genetic divergence in mtDNAs among natural populations of the higher basidiomycete Pleurotus ostreatus. Reciprocal crosses were made between compatible monokaryons with distinguishable mtDNA restriction fragment length polymorphisms (PFLPs). Almost all of the dikaryons produced by these crosses had mtDNA genotypes from one of the parental monokaryons. However, for dikaryons isolated from the junction-zone of crossed monokaryons, recombinant mitochondrial genomes commonly appeared. These results showed that P. ostreatus mtDNA can be inherited biparentally, via mtDNA recombination, as well as uniparentally. Further, it was suggested that mtDNA recombination may be an important source of variation in mitochondrial genomes among natural populations of P. ostreatus. Received: 4 June / 14 August 1996     

Maternal inheritance of mitochondrial DNA in sexual crosses of Pythium sylvaticumm

Summary Fifty single oospore progeny from crosses between opposite mating types of Pythium sylvaticum that contained polymorphisms in mitochondrial DNA (mtDNA) for HindIII restriction sites were analyzed for patterns of mitochondrial inheritance. All progeny retained the morphological form of the oogonial parental type; the antheridial form or recombinant forms of parental mtDNA were not detected. With the techniques used, other forms of mtDNA would have been detected it they had comprised 8% or more of the mitochondrial population.     

The mitochondrial plasmid of the true slime mold <Emphasis Type="Italic">Physarum polycephalum</Emphasis> bypasses uniparental inheritance by promoting mitochondrial fusion

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF⁺ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF^– strain KM88. Of the three mF^– × mF⁺ crosses, only KM88 × JE8 displayed complete uniparental inheritance. However, in KM88 × TU111 and KM88 × NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.The first two authors have equally contributed to this work     

The mating type-specific homeodomain genes <Emphasis Type="Italic">SXI1α</Emphasis> and <Emphasis Type="Italic">SXI2a</Emphasis> coordinately control uniparental mitochondrial inheritance in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

In the great majority of sexual eukaryotes, mitochondrial genomes are inherited almost exclusively from a single parent. While many hypotheses have been proposed to explain this phenomenon, very little is known about the genetic elements controlling uniparental mitochondria inheritance. In the bipolar, isogamous basidiomycete yeast Cryptococcus neoformans, progeny from crosses between strains of mating type a (MATa) and mating type α (MATα) typically inherit mitochondrial DNA (mtDNA) from the MATa parent. We recently demonstrated that a mating type α (MATα)-specific gene SXI1α, controls mitochondrial inheritance in C. neoformans. Here, we show that another homeodomain gene SXI2a in the alternative mating type MATa is also required for uniparental mtDNA inheritance in this fungus. Disruption of SXI2a resulted in biparental mtDNA inheritance in the zygote population with significant numbers of progeny inheriting mtDNA from the MATa parent, the MATα parent, and both the MATa and the MATα parents. In addition, progeny from same-sex mating between MATα strains showed a biparental mitochondrial inheritance pattern. Our results suggest that SXI1α and SXI2a coordinately control uniparental mitochondrial inheritance in C. neoformans.     

Mitochondrial inheritance in haploid × non-haploid crosses in Cryptococcus neoformans

In the basidiomycetous yeast Cryptococcus neoformans, fusants and meiotic progeny from haploid–haploid (HH) crosses between strains of mating type a (MAT a) and mating type alpha (MATα) typically inherit mitochondrial DNA (mtDNA) from the MAT a parent. In this study, we investigated the mtDNA inheritance pattern in haploid × non-haploid crosses. A total of 420 meiotic progeny and 173 fusants were obtained from five crosses and analyzed for two polymorphic mitochondrial markers. The percentage of meiotic progeny and fusants inheriting mtDNA from MATα or MATα/α parents ranged from 8 to 50%. The leakage was significantly greater than those observed in HH crosses, indicating that mtDNA inheritance is not uniparental in haploid × non-haploid crosses in C. neoformans. In addition, mtDNA leakage in the fusants, but not the meiotic progeny, of the MATα/α × MAT a cross was significantly higher than that in the MAT a/a × MATα cross, suggesting that the diploid parents with different mating types contribute differently in determining fusant mtDNA genotype in these crosses. Flow cytometry analysis revealed that meiotic progeny population of each cross was of mixed ploidy while the ploidy level of the selected fusants ranged from diploid to triploid.     

An ameiotic mutant of Coprinus cinereus halted prior to pre-meiotic S-phase

Summary Five Coprinus cinereus monokaryons were isolated which bore a dominant mutation designated Mar. Dikaryons formed by mating Mar-bearing monokaryons with normal monokaryons produced aborted fruiting bodies in which the basidia never underwent karyogamy. The Mar mutation probably prevented pre-meiotic DNA replication; extracts of Mar-bearing dikaryons harvested at a stage equivalent to pre-meiotiv S-phase caused little net DNA synthesis in DNA polymerase assays. There was no marked incorporation of 32p into DNA (and low incorporation of 32p into RNA) of cap tissue from Mar-bearing fruiting bodies at a stage equivalent to pre-meiotic S-phase. The aborted fruiting bodies were similar to those resulting on normal cultures following treatment prior to S-phase with cycloheximide, or continuous light at 35 °C. In contrast to normal C. cinereus monokaryons, no Mar-bearing monokaryon formed fruiting bodies when subjected to nutritional stress.     

Maternal transmission of mitochondrial DNA in interspecific hybrids of Populus

Summary Restriction fragment analysis was conducted to investigate the mode of inheritance of mitochondrial (mt) DNA in F₁ progeny of two P. deltoides x P. deltoides, three P. deltoides x P. nigra, and two P. deltoides x P. maximowiczii controlled crosses, and in Populus x canadensis by using 16 restriction endonucleases and two heterologous probes of cloned mtDNA fragments of maize. Five restriction fragment length polymorphisms (RFLPs) of mtDNA differentiated P. deltoides from P. nigra, whereas three RFLPs of mtDNA separated P. deltoides from P. maximowiczii. In all cases, F₁ progeny of P. deltoides x P. nigra, and P. deltoides x P. maximowiczii, crosses had mtDNA restriction fragments of only their maternal P. deltoides parents. P. x canadensis had mtDNA restriction fragments of only P. deltoides. F₁ progeny of intraspecific P. deltoides crosses also had the same mtDNA fragments as their maternal parent. The results clearly demonstrate uniparental-maternal inheritance of the mitochondrial genome in F₁ interspecific hybrids of P. deltoides with P. nigra and P. maximowiczii.     

Studies of mitochondrial morphology and DNA amount in the rice egg cell

In plant vegetative cells, mitochondria are usually small and grain-shaped. In contrast, unusually shaped giant mitochondria (large cup-shaped or long stretched-rod-shaped) appear in the egg cells of geranium, maize, Ipomoea nil, and bracken. In this study, to characterize egg cell mitochondria in rice, we used nonenzymatic manual dissection to isolate unfertilized egg cells of rice and observed the egg cell mitochondria and mitochondrial DNA (mtDNA) simultaneously. These observations showed that the mitochondria in the rice egg cell are small and grain-shaped, unlike the mitochondria in geranium, maize, I. nil, and bracken. Double staining of mitochondria by MitoTracker and mtDNA by SYBR Green I showed that mitochondria in the rice egg cell have a large amount of mtDNA compared with the rice root protoplast. We also used real-time PCR analysis to quantify the mtDNA amount in the rice egg cell. We quantified the copy numbers of four mitochondrial genes per single rice egg cell and rice leaf protoplast. Real-time PCR analysis revealed that the egg cell has more than ten times more copy numbers of all of four genes encoded in the mitochondrial genome compared with the leaf protoplast.     

Mitochondrial genetics of Coprinus: Recombination of mitochondrial genomes

Summary The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [acu-10] causing cytochrome aa ₃ deficiency and[cap-1.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons.     

Elimination of mitochondrial mutations by sexual reproduction: two Podospora anserina mitochondrial mutants yield only wild-type progeny when mated

In order to understand the transmission of mitochondrial mutations in sexual crosses of Podospora, we attempted to create compatible strains with defined mitochondrial mutations. A previously characterized mutant, Mn19, with a bipartite mitochondrial genome, served as the fertilizing parent in a cross with a mitochondrial deletion mutant, αΔ5. Characterization of the deletion mutant is reported here. All six of the monokaryotic progeny isolated had neither parental defect but instead appeared to have inherited wild-type mitochondrial DNA. One of the progeny had a mitochondrial plasmid derived from intramolecular recombination between an 11-bp repeated mitochondrial sequence. Subsequent analysis using the polymerase chain reaction (PCR) identified rare undeleted wild-type mtDNA sequences in the maternal parent. The uniform inheritance of wild-type mitochondrial DNA suggests either an aggressive repair mechanism or else selective amplification and transmission of rare wild-type mtDNA molecules. Received: 12 December 1995 / 6 May 1996     

Constitutive homologous recombination between mitochondrial DNA and a linear mitochondrial plasmid in Physarum polycephalum

Summary In one particular myxamoebal strain (NG7; mF⁺) of Physarum polycephalum, a linear mitochondrial plasmid (mF plasmid) which promotes mitochondrial fusion has been identified. A mating between mF^- strains, that do not carry the mF plasmid, resulted in uniparental inheritance of the mtDNA. In matings between mF⁺ and mF^- strains a recombination occurred between the mtDNA and the mF plasmid, and recombinant mtDNA was generated with the end of the mF plasmid as its ends. The DNA sequences of the recombination site in the mtDNA and the mF plasmid, and of the recombinant mtDNA, revealed that the mF plasmid had a 473-bp sequence that was identical to, but slightly shorter than, a 477-bp sequence of the mtDNA. This so-called identical sequence was found at the junction between unique sequences of the mF plasmid and the mtDNA in the recombinant mtDNA. Thus, the recombination between the mtDNA and the mF plasmid was due to reciprocal crossing-over at the identical sequence.     

New alleles of mgm1: a gene encoding a protein with a GTP-binding domain realted to dynamin

Three previously described genes that affect baker's yeast (Saccharomyces cerevisiae) mitochondrial DNA (mtDNA) or mitochondrial RNA, tpm2-1, mnal-1, and mgml-1, are shown to be alleles of the same gene. This report demonstrates that tpm2-1 does not affect recombination of mtDNA. Therefore, there is no evidence that this dynamin-like protein is involved in movement of mtDNA within a cell.     

Segregant-defective heterokaryons of Candida albicans

Summary Heterokaryons (hets) of the asexual, pathogenic yeast Candida albicans obtained by fusing protoplasts of complementing auxotrophic strains generate large numbers of parental-type auxotrophic monokaryons by random assortment of single nuclei into blastospores, and smaller numbers of monokaryons bearing hybrid nuclei formed through either karyogamy or the transfer of genetic material from one het nucleus to another. Het populations grown at 30 °C or 37 °C contain high frequencies (approx. 5%–10%) of two kinds of stable variants peculiar specifically for segregation of parental-type monokaryons: NS variants produce inviable auxotrophic monokaryons of one or both parental classes while AT variants yield parental-type monokaryons which grow very slowly. Variant frequencies are not affected by the wild-type strain background of hets, or the auxotrophies used to force heterokaryosis. However, both kinds of variants are induced by growth at 25 °C or by treatments with certain chemical or physical metabolic inhibitors. Evidence is presented that variant nuclei of independent origins carry different nutritionally irreparable recessive lethal (NS) or debilitating (AT) defects acquired in the course of actual or potential internuclear transfers of genetic material within het cells. The high incidence of variants, therefore, indicates considerable intrinsic genetic instability among het nuclei. Significances of these observations for parasexual genetic analyses of C. albicans and other yeasts through protoplast fusions are considered.     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs

Strict maternal inheritance of mitochondrial (mt) DNA is believed to be the rule in most eukaryotic organisms because of exclusion of paternal mitochondria from the egg cytoplasm during fertilization. In honeybees, polyspermic fertilization occurs, and many spermatozoa, including their mitochondria-rich flagellum, can completely penetrate the egg, thus allowing for a possibly high paternal leakage. In order to identify paternal mtDNA in honeybee eggs, restriction fragment length polymorphisms (RFLP) of different subspecies were used. Total DNA extracts of different developmental stages of an Apis mellifera carnica x Apis mellifera capensis hybrid brood were tested with a radioactively-labelled diagnostic mtDNA probe. Densitograms of autoradiographs indicated that the male contribution represents up to 27% of the total mitochondrial DNA in the fertilized eggs 12 h after oviposition. In subsequent developmental stages the portion of paternal mtDNA slowly decreased until hatching of the larvae when only traces were found. Although rapid disintegration of paternal mtDNA does not occur, the initially high paternal mitochondrial contribution is not maintained in the adult animal.     

Rapid disappearance of one parental mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum

Summary Five haploid amoebal strains of the myxomycete Physarum polycephalum, each with a distinct mitochondrial genotype, were crossed in all pairwise combinations. The mitochondrial genotype in the diploid plasmodia resulting from these isogamous matings were found to be transmitted uniparentally. This uniparental inheritance could be arranged in a dominant hierarchical order. Time-course analysis of the presence of mitochondrial genotypes in the zygotes and young developing plasmodial genotypes is virtually completed during the first two nuclear cycles in the zygote/differentiating plasmodium. To our knowledge this is the first report indicating an active mechanism involving the degradation of mitochondrial genomes in sexual crosses.     

Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts

We have isolated the total cellular DNA from the cultured diploid fibroblasts of a six-member, three-generation human family. Using a specific radioactive probe for mitochondrial (mt) sequences we have identified new polymorphic variants in this family for the Hhal restriction endonuclease cleavage pattern of the mtDNA. The inheritance of these cleavage patterns verifies the maternal inheritance of mtDNA through all three generations.     

High-frequency inter-parental recombination between mitochondrial genomes of rice cybrids

Analyzing more than 100 independent rice cybrids, we found evidence for inter-molecular recombination between parental mitochondrial genomes occurring at high frequency soon after protoplast fusion. The structure of the region around the atp6 gene showed extensive polymorphism among Indica (MTC-5A), Japonica (Nipponbare), and wild abortive (IR58024A) mitochondrial genomes. Recombination between the mitochondrial genomes of IR58024A and MTC-5A around the atp6 gene was detected by Southern-blot analysis of cybrid plants. Such recombinant mitochondrial molecules were also cloned from IR58024A/Nipponbare cybrid callus. PCR analysis around the atp6 gene demonstrated that inter-parental recombination occurs in practically all cybrid calli within 2 weeks after protoplast fusion. At this point, parental and recombinant mitochondrial genomes coexisted within the callus. Over the course of further cultivation, however, mitochondrial genome diversity decreased as parental and/or recombinant genomes segregated out.     

Segregation and transmission of mitochondrial markers in fusion products of the asporogenous yeast Torulopsis glabrata

Summary Using a protoplast fusion technique we have been able to locate to the mitochondrial genome of the asporogenous yeast Torulopsis glabrata mutations conferring resistance to oligomycin, antimycin and diuron. When two strains differing in the size of their mtDNAs were fused the mitochondrial markers from the parent with the larger mtDNA (71–91) were transmitted predominantly among the fusion products. Both genetical and physical evidence support the occurrence of recombination in T. glabrata mitochondrial genome. Segregation of the mitochondrial genome appears to take place before the separation of the first bud from the fusion product.