mtDNA Recombination Research Articles

Genetic exchange and recombination in populations of the root‐infecting fungus Armillaria gallica

Genetic individuals, or genets, of Armillaria and other root‐infecting basidiomycetes are usually found in discrete patches that often include the root systems of several adjacent trees. Each diploid individual is thought to arise in an unique mating event and then grow vegetatively in an expanding territory over a long period of time. Our objective in this study was to describe the population from which such genetic individuals are drawn. In a sample including 274 collections representing 121 genetic individuals of A. gallica (synonym A. bulbosa) from two sites in each of four regions of eastern North America, genotype frequencies at seven nuclear loci were not significantly different from Hardy‐Weinberg expectations. Furthermore, allele frequencies at the seven loci were not significantly different between regions. Additional allelic data from four non‐contiguous regions of mitochondrial DNA showed little or no population subdivision over the four regions. Analysis of the distribution of multilocus mtDNA haplotypes revealed some clonal transmission of mtDNAs between genets and nonrandom mating within sites. Despite the sharing of mtDNA types by some individuals, the overall sample contained a high level of genotypic diversity. The apparent linkage equilibrium between some pairs of loci and the high level of phylogenetic inconsistency among all four loci suggest the occurrence heteroplasmy and recombination among mtDNAs of A. gallica in nature. In laboratory matings of two haploid strains with different mtDNA types, a low frequency of recombination in mtDNA was detected.

Genetic exchange and recombination in populations of the root-infecting fungus Armillaria gallica.

Genetic individuals, or genets, of Armillaria and other root-infecting basidiomycetes are usually found in discrete patches that often include the root systems of several adjacent trees. Each diploid individual is thought to arise in an unique mating event and then grow vegetatively in an expanding territory over a long period of time. Our objective in this study was to describe the population from which such genetic individuals are drawn. In a sample including 274 collections representing 121 genetic individuals of A. gallica (synonym A. bulbosa) from two sites in each of four regions of eastern North America, genotype frequencies at seven nuclear loci were not significantly different from Hardy-Weinberg expectations. Furthermore, allele frequencies at the seven loci were not significantly different between regions. Additional allelic data from four non-contiguous regions of mitochondrial DNA showed little or no population subdivision over the four regions. Analysis of the distribution of multilocus mtDNA haplotypes revealed some clonal transmission of mtDNAs between genets and nonrandom mating within sites. Despite the sharing of mtDNA types by some individuals, the overall sample contained a high level of genotypic diversity. The apparent linkage equilibrium between some pairs of loci and the high level of phylogenetic inconsistency among all four loci suggest the occurrence heteroplasmy and recombination among mtDNAs of A. gallica in nature. In laboratory matings of two haploid strains with different mtDNA types, a low frequency of recombination in mtDNA was detected.

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

Three previously described genes that affect baker's yeast (Saccharomyces cerevisiae) mitochondrial DNA (mtDNA) or mitochondrial RNA, tpm2-1, mna1-1, and mgm-1-1, 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.

Diplotaxis catholica + Brassica juncea somatic hybrids: molecular and cytogenetic characterization

Intergeneric somatic hybrids Diplotaxis catholica (2n=18) + Brassica juncea (2n=36) were produced by fusing mesophyll protoplasts of the former and hypocotyl protoplasts of the latter using polyethylene glycol. Out of 52 somatic embryos, 24 produced plants of intermediate morphology. Cytological analysis of 16 plants indicated that 15 were symmetric hybrids carrying 54 chromosomes, the sum of the parental chromosome numbers. One hybrid was asymmetric with 45 chromosomes. Nuclear hybridity of five putative hybrids was confirmed by the Southern hybridization pattern of full length 18s-25s wheat nuclear rDNA probe which revealed the presence of Hind III fragments characteristic of both the parental species. The hybridization pattern of mitochondria specific gene probe cox I indicated that three of the hybrids carried B. juncea mitochondria and one carried mitochondria of D. catholica. Presence of novel 3.5 kb Hind III and 4.8 kb Bgl II fragments suggested the occurrence of mtDNA recombination in one of the hybrids. The hybrids were pollen sterile. However, seeds were obtained from most of the hybrids by back crossing with B. juncea.

Restriction fragment length polymorphism (RFLP) analysis provides evidence for a high degree of homology of mitochondrial DNAs from rat hepatomas versus normal rat livers.

Where differences have been reported between tumor and normal mitochondrial DNA (mtDNA), they have generally involved limited modifications of the genome (Taira et al., Nucleic Acids Res. 11:1635, 1983; Shay and Werbin, Mutat. Res. 186:149, 1987). However, Corral et al. (Nucleic Acids Res. 16:10935, 1988; 17:5191, 1989) observed recombination between cytochrome oxidase subunit I (COI) and NADH dehydrogenase subunit 6 (ND6), two genes normally on opposite sides of the circular mitochondrial genome. In rat hepatoma mtDNA COI and ND6 were reported to be separated by only 230 base pairs (Corral et al., 1988, 1989). We have performed RFLP analysis on mtDNA from normal rat livers and rat hepatomas, using COI and ND6 probes. Additional experiments compared end-labeled DNA fragments produced by EcoRI and HindIII digestion of mtDNA. These studies failed to provide any evidence for genetic recombination in rat hepatoma mtDNA, even in the same cell line used by Corral et al. Rather, they support the conclusion that mtDNA from tumor and normal tissues exhibits a low degree of heterogeneity.

PIF1: a DNA helicase in yeast mitochondria.

The PIF1 gene is involved in repair and recombination of mitochondrial DNA (mtDNA). In this study, the PIF1 gene product, which cannot be identified in normal yeast cells, has been overproduced from the GALI promoter to detectable protein levels. Location of PIF1 in mitochondria has been shown by immunoelectron microscopy and in vivo import experiments using ts mas1 mutants deficient in the mitochondrial matrix-localized processing protease. Overproduction of PIF1 protein in pif1 mutants restores mtDNA recombination proficiency but is toxic to yeast cells as observed by slower growth. The overproduced PIF1 protein, which is firmly associated with insoluble mitochondrial structures, has been partially purified in a mitochondrial nuclease deficient nuc1 strain by a procedure including solubilization by urea and renaturation by dialysis at alkaline pH. PIF1 is a single-stranded (ss) DNA-dependent ATPase and a DNA helicase which unwinds partially DNA duplexes in a 5' to 3' direction with respect to the ss DNA on which it binds first.

MITOCHONDRIAL DNA POLYMORPHISM AND CYTOPLASMIC INCOMPATIBILITY IN NATURAL POPULATIONS OF DROSOPHILA SIMULANS.

The study of restriction polymorphism of animal mitochondrial DNA (mtDNA) in natural populations occupies an important place in the field of phylogeography. Populations are generally expected to show a greater degree of genetic subdivision for mtDNA variation than for allozymes (Birky et al., 1989). This is a natural outcome of the transmission dynamics of mtDNA. Because of uniparental (maternal) transmission, there is a lower effective copy number of mitochondrial genes in a population relative to nuclear genes. This leaves mtDNA variation less susceptible to recurrent migration and more susceptible to population bottlenecks. As a result, analysis of mtDNA polymorphism distributions, coupled with genealogical reconstruction (which is simplified by the lack of recombination in mtDNA) will yield information on population history that is less likely than nuclear variation to be obscured by recurrent gene flow. However, population structure of mtDNA variation can, in theory, be influenced by other processes. One possibility is natural selection, as some interactive effects of chromosomal and mitochondrial transmission have been suggested in Drosophila (Clark, 1985; Clark and Lyckegaard, 1988; MacRae and Anderson, 1988). Another is asymmetric fertility in crosses between closely related species or diverged populations. If one of the two reciprocal hybridizations has a higher fertility, then the cytoplasm, and thereby the mtDNA, of the maternal parent in the favored cross will increase in frequency. Hoffmann et al. (1986) and Hoffmann and Turelli (1988) described a case of asymmetric fertility within and between sympatric populations ofcontinental Drosophila simulans. They identified two incompatibility types in which one type (designated R) is associated with a Wolbachia-like microorganism in the maternal cytoplasm (Binnington and Hoffmann, 1989). The other type (designated W) does not harbor the microorganism. Very few progeny are produced when W females are crossed to R males. The reciprocal cross

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56-4.48 kg/ha (0.5-4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.

Sensitivity of intergenic regions of yeast mitochondrial DNA to single-strand-specific nucleases

The reactivity of mitochondrial DNA (mtDNA) sequences from Torulopsis glabrata and Saccharomyces cerevisiae towards single-strand-specific nucleases has been examined. AT-rich stretches located in intergenic sequences from both yeasts were cleaved by nucleases when the sequences were contained in supercoiled plasmid DNA. In particular ori/rep sequences from the mtDNA of S. cerevisiae were shown to be sensitive to the single-strand-specific nucleases. The locations of the sensitive sites were related to the organisation of the sequence domains of ori/rep and the superhelicity of the DNA, as well as the presence of particular sequences. It is proposed that distortions of the DNA duplex could be generated in mtDNA molecules in vivo and that these distortions may provide a substrate for enzymes involved in transmission, recombination and/or transcription of mtDNA.

Preferential amplification of mitochondrial DNA fragments in somatic hybrids of the Gramineae

Somatic hybrid cell lines of Pennisetum americanum + Panicum maximum, and of Pennisetum americanum + Saccharum officinarum display unique mitochondrial DNA (mtDNA) restriction patterns suggestive of mitochondrial fusion and recombination. Apparent recombinant fragments of the hybrids were recovered, cloned, and hybridized to parental and somatic hybrid mtDNAs. In each somatic hybrid, “novel” fragments were found to be present at low copy number in one or both of the parental mtDNAs, and amplified 15–30 times in the hybrids. In pearl millet-sugarcane somatic hybrid cells, the amplification does not appear to involve enhanced recombination. The presumably amplified restriction fragment of the pearl millet-Guinea grass somatic hybrids is a junction fragment of a repeat, present in low copy number in both parents, and in high copy number in the hybrids. Thus protoplast and probable mitochondrial fusion results in a marked shift in the direction of mtDNA recombination events. We conclude that amplification of parental mtDNA fragments is a common event in somatic hybrid cells of these Gramineae.

Restoration of male fertileNicotiana by fusion of protoplasts derived from two different cytoplasmic male-sterile cybrids

Using the 'donor-recipient' protoplast-fusion technique, we have recently constructed several alloplasmic-like lines ofNicotiana in which the original cytoplasms (or part of them) of eitherN. tabacum orN. sylvestris were replaced respectively, either byN. undulata or byN. bigelovii cytoplasms. These cybridizations resulted in two kinds of cytoplasmic male-sterile (CMS) cybrid plants:N. tabacum withN. undulata-like cytoplasm andN. sylvestris withN. bigelovii-like cytoplasm. Fusion of protoplasts, derived from the above two CMS types, by the 'donor-recipient' technique, lead to the recovery of 21 cybrid calli. One of these regenerated a cybrid with fertile pollen but having shortened filaments and slighly tappered anthers. Self pollination of the latter cybrid resulted in a second generation progeny having almost normal filaments and anthers. Further selfings produced a third generation in which numerous plants had normal stamens and fertile pollen. Mitochondrial DNA (mtDNA) analysis of second and third generation progenies revealed a novel pattern which differed from each of the parental CMS cybrids and also from the mtDNA of normal, male-fertileNicotiana species. The results suggest that mtDNA recombination between different types of CMS can lead to restoration of male-fertility.

Absence of extensive recombination between inter- and intraspecies mitochondrial DNA in mammalian cells

Recombination of mammalian mitochondrial DNA (mtDNA) was examined using mouse X rat somatic cell hybrid clones and rat cybrid clones. The mouse X rat hybrids were isolated by fusion of chloramphenicol-sensitive (CAP s) mouse and CAP-resistant (CAP r) rat cells. The rat cybrids were isolated by fusion of rat cells with type B mtDNA and enucleated cells with type A mtDNA. Genetic and physical analyses showed that the mtDNAs of the hybrids and cybrids were simple mixtures of the two parental mtDNAs except in the following two cases: One was subclone H29 of mouse X rat hybrids, which was CAP r even though mtDNA from the CAP s mouse parent was predominantly retained. The other was rat cybrid subclones, Y1224 and -61, which showed specific loss of one Hinf I fragment of type B mtDNA, B10. These observations suggest that, in contrast to the case with plant mtDNA, recombination of mammalian mtDNA occurs rarely, if at all.

The organization of repeating units in mitochondrial DNA from yeast petite mutants

We have reinvestigated the linkage orientation of repeating units in mtDNAs of yeast ρ(-) petite mutants containing an inverted duplication. All five petite mtDNAs studied contain a continuous segment of wild-type mtDNA, part of which is duplicated and present in inverted form in the repeat. We show by restriction enzyme analysis that the non-duplicated segments between the inverted duplications are present in random orientation in all five petite mtDNAs. There is no segregation of sub-types with unique orientation. We attribute this to the high rate of intramolecular recombination between the inverted duplications. The results provide additional evidence for the high rate of recombination of yeast mtDNA even in haploid ρ(-) petite cells.We conclude that only two types of stable sequence organization exist in petite mtDNA: petites without an inverted duplication have repeats linked in straight head-to-tail arrangement (abcabc); petites with an inverted duplication have repeats in which the non-duplicated segments are present in random orientation.