High-rate spontaneous reversion to cytoplasmic male sterility in sugar beet: a characterization of the mitochondrial genomes

Summary Among the fertile sugar beet lines with nuclear sterility maintenance genes, rf, in a homozygous recessive state, sublines capable of reverting spontaneously at a high rate to sterility were identified. Of 24 related fertile sublines studied, 6 were found to spontaneously revert to sterility with a frequency of about 19%. Genetic analysis confirmed the cytoplasmic nature of spontaneously arising sterility. Reversion to sterility in these sublines was accompanied by alterations in the mitochondrial genome structure: loss of the autonomously replicating minicircle c (1.3 kb) and changes in the restriction patterns of high-molecular-weight mitochondrial DNA (mtDNA). Southern hybridixation analysis with cloned minicircle c as a probe revealed no integration of this DNA molecule into the main mitochondrial and nuclear genomes of the revertants. Comparative BamHI and EcoRI restriction analysis of the mtDNA from the sterile revertants and fertile parental subline showed that the spontaneous reversion is accompanied by extensive genomic rearrangement. Southern blot analysis with cloned -subunit of F₁-ATPase (atpA) and cytochrome c oxidase subunit II (COX II) genes as probes indicated that the changes in mtDNA accompanying spontaneous reversion to sterility involved these regions. The mitochondrial genomes of the spontaneous revertants and the sterile analogue were shown to be identical.     

Patterns of nucleotide change in mitochondrial ribosomal RNA genes and the phylogeny of piranhas

The patterns and rates of nucleotide substitution in mitochondrial ribosomal RNA genes are described and applied in a phylogenetic analysis of fishes of the subfamily Serrasalminae (Teleostei, Characiformes, Characidae). Fragments of 345 bp of the 12S and 535 bp of the 16S genes were sequenced for 37 taxa representing all but three genera in the subfamily. Secondary-structure models based on comparative sequence analysis were derived to characterize the pattern of change among paired and unpaired nucleotides, forming stem and loop regions, respectively. Base compositional biases were in the direction of A-rich loops and G-rich stems. Ninety-five percent of substitutions in stem regions were compensatory mutations, suggesting that selection for maintenance of base pairing is strong and that independence among characters cannot be assumed in phylogenetic analyses of stem characters. The relative rate of nucleotide substitution was similar in both fragments sequenced but higher in loop than in stem regions. In both genes, C-T transitions were the most common type of change, and overall transitions outnumbered transversions by a factor of two in 16S and four in 12S. Phylogenetic analysis of the mitochondrial DNA sequences suggests that a clade formed by the generaPiaractus, Colossoma, andMylossoma is the sister group to all other serrasalmins and that the generaMyleus, Serrasalmus, andPristobrycon are paraphyletic. A previous hypothesis concerning relationships for the serrasalmins, based on morphological evidence, is not supported by the molecular data. However, phylogenetic analysis of host-specific helminth parasites and cytogenetic data support the phylogeny of the Serrasalminae obtained in this study and provide evidence for coevolution between helminth parasites and their fish hosts.     

The complete mitochondrial DNA (mtDNA) of the donkey and mtDNA comparisons among four closely related mammalian species-pairs

The nucleotide sequence of the complete mitochondrial genome of the donkey, Equus asinus, was determined. The length of the molecule is 16,670 bp. The length, however, is not absolute due to pronounced heteroplasmy caused by variable numbers of two types of repetitive motifs in the control region. The sequence of the repeats is (a) 5′-CACACCCA and (b) 5′-TGCGCGCA, respectively. The order of (a) and (b) can be expressed as {n[2(a)+(b)]+m(a)}. In 32 different clones analyzed the number of n and m ranged from 0 to 9 and 1 to 7. The two rRNA genes, the 13 peptide-coding genes, and the 22 tRNA genes of the donkey and the horse, Equus caballus, were compared in detail. Total nucleotide difference outside the control region was 6.9%. Nucleotide difference between peptide-coding genes ranged from 6.4% to 9.4% with a mean of 8.0%. In the inferred protein sequences of the 13 peptide-coding genes the amino acid difference was 0.2–8.8%, and the mean for the 13 concatenated amino acid sequences was 1.9%. In the 22 tRNA genes, the mean difference was 3.5%, and that in the two rRNA genes was 4.1%. The mtDNA differences between the donkey and the horse suggest that the evolutionary separation of the two species occurred ≈9 million years ago. Analyses of differences among the mtDNAs of three other species-pairs, harbor seal/grey seal, fin whale/blue whale, and Homo/common chimpanzee, showed that the relative evolutionary rate of individual peptide-coding genes varies among different species-pairs and modes of comparison. The findings show that the superimposition of sequence data of one lineage for resolving and dating evolutionary divergences of other lineages should be performed with caution unless based on comprehensive data. Received: 15 October 1995 / Accepted: 15 April 1996     

comparison between the complete mitochondrial DNA sequences ofHomo and the common chimpanzee based on nonchimeric sequences

The complete mitochondrial DNA (mtDNA) molecules ofHomo and of the common chimpanzee were sequenced. Each sequence was established from tissue of one individual and thus nonchimeric. Both sequences were assembled in their entirety from natural (not PCR amplified) clones. Comparison with sequences in the literature identified the chimpanzee specimen asPan troglodytes verus, the West African variety of the species. The nucleotide difference between the complete human and chimpanzee sequences is 8.9%. The difference between the control regions of the two sequences is 13.9% and that between the remaining portions of the sequences 8.5%. The mean amino acid difference between the inferred products of the 13 peptide-coding genes is 4.4%. Sequences of the complete control regions, the complete 12S rRNA genes, the complete cytochromeb genes, and portions of the NADH4 and NADH5 genes of two other chimpanzee specimens showed that they were similar but strikingly different from the same regions of the completely sequenced molecule fromPan troglodytes verus. The two specimens were identified asPan troglodytes troglodytes, the Central African variety of the common chimpanzee.     

The biallelica mating type locus ofUstilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor

Thea mating type locus ofUstilago maydis contains the structural genes for a pheromone-based cell recognition system that governs fusion of haploid cells. The locus exists in two alleles, termeda1 anda2. We have completed the analysis of the nucleotide sequences unique toa1 anda2. Within these dissimilar regions we find two short patches of DNA sequence similarity. Interestingly, one of these segments corresponds to the transcribed region of thea1 pheromone precursor. As a result of multiple nucleotide exchanges this sequence does not code for a functional product. The existence of a second pheromone gene in thea2 allele suggests that the present locus had a multiallelic ancestor. In addition, we describe the presence of two additional genes in thea2 allele. We have investigated the role of these genes during mating and pathogenic development and speculate that they might affect mitochondrial inheritance.     

Accumulation of mitochondrial DNA deletions in myotubes cultured from muscles of patients with mitochondrial myopathies

 Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percent- age of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA in the first patient’s cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation. Received: 16 April 1996/Accepted: 29 July 1996     

Studies on the development and three-dimensional reconstruction of giant mitochondria and their nuclei in egg cells ofPelargonium zonale Ait.

Summary The preferential development of giant mitochondria and their nuclei (nucleoids) in the egg cells ofPelargonium zonale Ait. during megasporogenesis and megagametogenesis was examined by fluorescence microscopy, after Technovit embedding and 4,6-diamidino-2-phenylindole (DAPI) staining, fluorimetry for DNA content, using a video-intensified microscope photon-counting system (VIMPICS), and by three-dimensional reconstruction of mitochondrial nuclei (mt-nuclei). Reproductive cells during the megaspore mother cell, meiosis, tetrad, and functioning megaspore stages contained many small mitochondria with characteristic, uniformly DAPI-stained mt-nuclei about 0.3 m in diameter, containing a small amount of DNA (0.3 Mbp). During formation of the 2-, 4-, and 8-nucleate embryo sac, mt-nuclei did not markedly change in shape or DNA content. When the embryo sac formed and differentiation of each cell began, mitochondria and their nuclei in the egg cell took on a small ring or string-like shape. Accompanying the maturation of the embryo sac, they underwent progressive enlargement and gradually altered to long thick strings, or stacks of concentric or half concentric rings. By flower opening, they have developed to an extremely large size. One of these stacks of mt-nuclei was reconstructed in three dimensions; each ring in the stack was cup- or plate-shaped; 5 to 10 rings made up the stack, though each remained discontinuous from the others. From serial sections, we counted 44 mitochondria in one egg cell. Fluorometry using VIMPICS revealed that DNA amount within the stacked mitochondrion increased to 40 times that of the megaspore mother cell stage; a single stack of mitochondria contained 340–1700 Mbp DNA; which means that one egg cell contains at least 15000 Mbp mt-DNA, a value greater than the cell-nuclear DNA content.     

Synthesis of hexaploid (AABBCC) somatic hybrids: a bridging material for transfer of ‘tour’ cytoplasmic male sterility to different Brassica species

Most of the alloplasmic cytoplasmic male sterility (CMS) systems are known to be associated with a number of floral abnormalities that result from nuclear-cytoplasmic incompatibilities. One such system, tour, which is derived from Brassica tournefortii, induces additional floral abnormalities and causes chlorosis in Brassica spp. While the restorer for this CMS has been reported to be present in B. napus, in B. juncea, where the abnormalities are more pronounced, no restorer has yet been identified. Rectification of these floral abnormalities through mitochondrial recombinations and chloroplast replacement might result in the improvement of this CMS system. As organelle recombinations can possibly be achieved only by somatic cell hybridization, fusion experiments were carried out between hygromycin-resistant B. juncea AABB carrying tour cytoplasm and phosphinotricin-resistant, normal B. oleracea CC to generate AABBCC hexaploid somatic hybrids. The presence of selectable marker genes facilitated the selection of hybrids in large numbers. The resulting hybrids showed wide variation in floral morphology and organelle composition. Regenerants with normal, male-sterile flowers having recombinant tour-or oleracea-type mitochondria and oleracea-type chloroplasts were obtained. Hybrids with male-fertile flowers were also obtained that had recombined tour mitochondria. The AABBCC hexaploid hybrids synthesized in the present study were successfully utilized as a bridging material for transferring variability in the organelle genome simultaneously to all the digenomic Brassica species, and all of these hybrids are now being stabilized through repeated backcrosses to the allopolyploid crop brassicas.     

Spatial distribution of cpDNA and mtDNA haplotypes in a hybrid zone betweenPinus pumila andP. parviflora var.pentaphylla (Pinaceae)

Pinus species exhibit paternal chloroplast inheritance and maternal mitochondrial inheritance. This independent inheritance of two cytoplasmic genomes provides an exceptional environment for discriminating female (seeds) and male (pollen) components of gene flow across hybridizing species. We obtained mitochondrial genetic markers diagnostic toP. parviflora var.pentaphylla andP. pumila by PCR amplification of the intron ofnad1 on mtDNA, and examined the spatial-distribution pattern of the mtDNA haplotypes in a hybrid zone betweenP. parviflora var.pentaphylla andP. pumila in the Tanigawa Mountains of Japan. These data, in conjunction with previous information on cpDNA haplotypes and needle morphology, revealed contrastive patterns of introgression of two cytoplasmic genomes. CpDNA introgression has occurred uni-directionally fromP. parviflora var.pentaphylla toP. pumila. Conversely, mtDNA introgression has occurred in the opposite direction, fromP. pumila toP. parviflora var.pentaphylla. Levels of introgression are roughly equivalent for cpDNA and mtDNA. The contrastive spatial distribution pattern of cpDNA and mtDNA haplotypes could be caused by differential movement of seeds and pollen for interspecific genetic exchange.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.