Evaluation of the Extent of Homologous Chloroplast DNA Sequences in the Mitochondrial Genome of Cowpea (Vigna unguiculata L.)

Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.     

Restriction polymorphism of mitochondrial DNA among Russian residents of Western Siberia]

Hereditary polymorphism of mitochondrial DNA is described for 5 restriction enzymes: BamHI, HindIII, PstI, PvuII and SacI in 60 Russian individuals living in Tomsk. 13 morphotypes of mitochondrial DNA were revealed as a whole for the enzymes analysed. Comparative analysis of mitochondrial DNA morphs and morphotypes with the literature data was conducted.     

Calorie restriction, oxidative stress and longevity

Studies on the relationship between oxidative stress and ageing in different vertebrate species and in calorie-restricted animals are reviewed. Endogenous antioxidants inversely correlate with maximum longevity in animal species and experiments modifying levels of these antioxidants can increase survival and mean life span but not maximum life span (MLSP). The available evidence shows that long-living vertebrates consistently have low rates of mitochondrial free radical generation, as well as a low grade of fatty acid unsaturation on cellular membranes, which are two crucial factors determining their ageing rate. Oxidative damage to mitochondrial DNA is also lower in long-living vertebrates than in short-living vertebrates. Calorie restriction, the best described experimental strategy that consistently increases mean and maximum life span, also decreases mitochondrial reactive oxygen species (ROS) generation and oxidative damage to mitochondrial DNA. Recent data indicate that the decrease in mitochondrial ROS generation is due to protein restriction rather than to calorie restriction, and more specifically to dietary methionine restriction. Greater longevity would be partly achieved by a low rate of endogenous oxidative damage generation, but also by a macromolecular composition highly resistant to oxidative modification, as is the case for lipids and proteins.     

Estimation of the neutral mutation rate in a finite population from DNA sequence data

The sampling theory for the infinite site model taking into account the phylogenetic relationship between the alleles is developed for those cases in which two or three alleles are observed in the sample. From this theory a maximum likelihood estimate of θ = 4Nμ can be obtained. Unlike the maximum likelihood estimate of θ based on the infinite allele model or the number of segregating sites, this estimate of θ is a function of the frequencies of the alleles. This method is used to estimate θ for mitochondrial DNA in Drosophila melanogaster and D. virilis from data obtained by Shah and Langley (1979. Nature (London)281, 696–699) using restriction endonucleases.     

The mitochondrial genome is large and variable in a family of plants (cucurbitaceae)

The genome sizes of mitochondrial DNA from darkgrown (etiolated) shoots of several higher plants were determined by reassociation kinetics and restriction analysis. Kinetic complexities obtained from reassociation kinetics measured spectrophotometrically indicate a mitochondrial genome size of 1600 Md for muskmelon, 1000 Md for cucumber, 560 Md for zucchini squash and 220 Md for watermelon (four species in the cucurbit family), as well as 240 Md for pea and 320 Md for corn. The kinetic curves also reveal the presence (except in corn) of sequences of a few magadaltons of complexity, reiterated about 10-50 times and representing 5%- 10% of the DNA in each mitochondrial genome. Molecular weight summation of fragments resulting from digestion with restriction endonucleases Sal I and Kpn I give genome size estimates similar to those obtained from reassociation kinetics, except for muskmelon and cucumber, for which the large number of fragments of similar size limits our estimate to at least 500 Md. The number of mitochondrial genomes per diploid cell is estimated to be about 110 to 140 for muskmelon, zucchini and watermelon. We consider the possible evolutionary mechanisms by which the mitochondrial genome has grown within the cucurbit family and the possible reasons for the existance of a seven to eight-fold range in mitochondrial genome size among such closely related species.     

Mitochondrial DNA variations in Russian and Belorussian populations

The sequence of the first hypervariable segment (HVS-I) of mitochondrial DNA (mtDNA) was determined in 251 individuals from three eastern Slavonic populations, two Russian and one Belorussian. Within HVS-I, 78 polymorphic positions were revealed. Within-population diversity of HVS-I varies slightly among three samples; its estimates do not differ strongly from those for European populations. Haplotype diversity for three populations calculated in this study is 0.949; mean pairwise differences estimate is 3.59. To assign mtDNA sequences to major phylogenetic clusters, haplogroup-specific restriction polymorphisms were selectively typed in most samples. The haplogroup distribution in the total Eastern Slavonic sample is similar to that reported for the European sample. However, the separate consideration of three Slavonic samples reveals the complicated structure of the mitochondrial gene pool in the Eastern European area. Data of this study support the proposed model of the origin of modern Eastern Slavs, which implies the admixture of ancient Slavonic tribes with pre-Slavonic populations of Eastern Europe. These data should contribute to general studies of mitochondrial DNA variations in Europe.     

TESTS OF PHYLOGEOGRAPHIC MODELS WITH NUCLEAR AND MITOCHONDRIAL DNA SEQUENCE VARIATION IN THE STONE CRABS,MENIPPE ADINA AND MENIPPE MERCENARIA

Evolutionary relationships among stone crabs (Menippe) from the Gulf of Mexico and western Atlantic were investigated by comparisons of restriction sites within anonymous nuclear DNA sequences and nucleotide sequences of both mitochondrial and a duplicated nuclear form of the mitochondrial large subunit ribosomal RNA (LSrDNA) gene. A survey of over 100 restriction sites by Southern blot analysis with 10 anonymous nuclear DNA sequence probes failed to reveal any differences between Menippe adina and M. mercenaria. Sequence comparisons of both mitochondrial and nuclear forms of the LSrDNA gene also did not distinguish these species. Although both LSrDNA gene sequences were variable, some haplotypes were shared by the two species, implying either incomplete gene lineage sorting or introgressive hybridization. Based on molecular clock calibrations, we estimate that all of the observed mitochondrial LSrDNA sequences share a common ancestor between 1.5 and 2.7 million years before present (M.Y.B.P.). However, because identical sequences are shared by the two species, these data are also compatible with a more recent common ancestry. These findings conflict with a previously proposed biogeographic scenario for North American Menippe, which featured a relict hybrid zone on the Atlantic Coast. We suggest an alternative scenario based on relatively recent events and ongoing, rather than historical, gene flow.     

Breeding origin and migration pattern of dunlin (Calidris alpina) revealed by mitochondrial DNA analysis

The large-scale migration of birds has been studied extensively by recoveries of ringed birds. However, there is very little ringing data from the arctic breeding grounds of waders. Here, the migration pattern of the dunlin, Calidris alpina, is studied with population genetic markers, using haplotype frequencies to estimate the breeding origin of migrating and wintering populations. Polymerase chain reaction (PCR) and restriction analysis of DNA from the mitochondrial control region was used to study the breeding origins of morphologically similar winter populations in the western Palaearctic, and to describe the population structure of the dunlin during winter. Also migrating dunlin from various stopover sites in Europe, Africa and Asia, were analysed with respect to their mitochondrial DNA (mtDNA) haplotypes. The genetic markers clearly show that the dunlin has a parallel migration system, with populations breeding in the western Palaearctic wintering mainly in the western part of the wintering range, and dunlin populations breeding further east wintering further east. The results also show that the distance between breeding and wintering area increases eastwards in this region.     

De novo synthesis of DNA in human platelets

Platelets, incubated with radiolabeled thymidine and purified free of contaminating nucleated cells, were analyzed for their ability to synthesize DNA. The only DNA species isolated from these purified platelets was mitochondrial DNA. The CsCl gradient-purified platelet DNA was treated with the restriction endonucleases EcoRI, HindIII and HpaI yielding the expected pattern for human mitochondrial DNA. Nitrocellulose blots of the electrophoresed, restriction endonuclease-treated DNA were fluorographed. All of the DNA fragments generated by the restriction enzymes were labeled, indicating de novo synthesis. This was further substantiated by inhibition of DNA synthesis by ethidium bromide and 2',3'-dideoxythymidine. Platelet DNA appeared to become greatly fragmented after 4 to 7 days storage while all of the thymidine incorporated was observed in intact mitochondrial DNA. These results indicate a continuous degradation of platelet mitochondrial DNA with no apparent repair mechanism. The ability of platelets to synthesize DNA may be associated with the protein synthetic capacity of platelets previously described.     

Lupin chloroplast and mitochondrial tRNA populations and their genes

Transfer RNAs isolated from lupin chloroplasts and mitochondria were compared by two-dimensional gel electrophoresis. Twenty chloroplast and 24 mitochondrial tRNA species were identified. The saturation hybridization between lupin chloroplast DNA and 125I-labelled lupin chloroplast tRNAs pointed to the presence of about 34 tRNA genes in lupin chloroplast DNA. The number of mitochondrial tRNA genes estimated by the same method was about 30 genes. EcoRI restriction digest of lupin mitochondrial DNA probed with 32P-labelled lupin mitochondrial tRNAs revealed only a small number of positive restriction fragments. Some of these mitochondrial restriction fragments hybridized with 32P-labelled chloroplast tRNA.     

Genetic relationships among the shads (Alosa) revealed by mitochondrial DNA analysis

We surveyed restriction site differences in mitochondrial DNA. (mtDNA) among five species of shad ( Alosa ) from North America and Europe. Allis shad, Alosa alosa and twaite shad, Alosa fallax shared two divergent genotype groups, suggesting that the two forms are either a single species, or are distinct species that have hybridized. Phenetic and cladistic analyses of the relationships among the mitochondrial genotypes defined two groups of shad, corresponding to the subgenera, Alosa and Pomolobus . The mean estimated sequence divergence between the mtDNAs of these two groups of shad was 6.5%. Taken in conjunction with fossil data, this divergence estimate suggests that the rate of mtDNA divergence between the two subgenera has been almost 10-fold lower than the 'conventional' clock calibration for mtDNA.     

Nucleotide sequence of the mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR.

Yeast mitochondrial DNA-pBR322 recombinant DNA molecules known to contain tRNA genes from a tRNA rich region of the yeast genome were used as a source of DNA for restriction mapping and tRNA gene sequence analysis. We report here restriction maps of two segments of yeast mitochondrial DNA and the sequence of mitochondrial genes coding for tRNAglyGGR and tRNAvalGUR. Both genes are flanked by A + T rich DNA and neither has an intervening sequence nor codes for a 3' CCA end. The tRNA structures deduced from the genes have the usual cloverleaf structures and invariant nucleotides. This combination of DNA sequencing and restriction mapping has enabled us to determine that the tRNAvalGUR and a previously sequenced tRNA, the tRNApheUUY are transcribed from the same strand of DNA.     

Phylogenetic relationships of sorghum taxa inferred from mitochondrial DNA restriction fragment analysis.

To elucidate the evolutionary history and affinity of sorghum species, 41 sorghum taxa were analyzed using variability in mitochondrial DNA. Analysis of species relationships at the molecular level can provide additional data to supplement the existing classification based on morphological characters and may also furnish unexpected but useful information. Total DNA extracted from each of the sorghum accessions was digested with each of five restriction enzymes, BamHI, HindIII, EcoRI, EcoRV, and XbaI, and probed with five mitochondrial DNAs cloned from Sorghumbicolor. A total of 180 restriction fragments was detected by the 25 probe-enzyme combinations. Forty-three fragment bands were phylogenetically informative. Multiple correspondence analysis was performed to visualize associations among the accessions and suggested that section Eusorghum species may be divided into four groups, with Sorghumlaxiflorum (section Heterosorghum) and Sorghumnitidum (section Parasorghum) appearing as outliers. A phylogenetic tree was assembled from mitochondrial restriction fragment data. The taxa analyzed formed three major groups comprising section Heterosorghum (group I), section Parasorghum (group II), and all accessions in section Eusorghum (group III). Group III is further divided into four groups: (i) two sweet sorghums and shattercane; (ii) Sorghumhalepense, Sorghummiliaceum, Sorghumhewisonii, Sorghumaethiopicum, Sorghumverticilliflorum, and S. bicolor, including Sorghumsudanense (sudangrass), the Chinese Kaoliangs, and a number of commercial sorghum inbreds from the U.S.A.; (iii) Sorghumpropinquum; and (iv) Sorghumarundinaceum, Sorghumniloticum, Sorghumalmum, Sorghumcontroversum, and the Chinese material C-401 and 5-27. Results indicate that the analysis of fragmented mitochondrial DNA was diagnostic and useful in sorghum phylogenetic and taxonomic research at the species, subspecies, and race levels, and can complement results from those analyses using nuclear ribosomal DNA and chloroplast DNA that effectively distinguish taxa at species and genus levels. Key words : Sorghum, mitochondrial DNA, phylogeny, restriction fragment.     

The ribosomal RNA genes on Neurospora crassa mitochondrial DNA are adjacent.

Hybridization of separated 24 S and 17 S ribosomal RNA from Neurospora crassa mitochondrial ribosomes to restriction fragments of mitochondrial DNA leads to the conclusion that the large and small ribosomal RNA are adjacent on the restriction endonuclease cleavage map of the DNA. The distance between the two genes is estimated at 900 basepairs. This result is consistent with the existence of a ribosomal precursor RNA in N. crassa mitochondria and is in contrast to the situation in yeast, where the ribosomal genes are far apart on the mitochondrial DNA. The position of the ribosomal RNA genes on the cleavage map of N. crassa mtDNA provides a start for ordering the Hind III restriction fragments.     

Mapping and cloning of Neurospora crassa mitochondrial transfer RNA genes.

We have obtained collections of recombinant Escherichia coli plasmids containing restriction fragments of Neurospora crassa mitochondrial DNA cloned into pBR322. By hybridization of 32P end-labeled total mitochondrial tRNAs and seven different purified tRNAs to restriction digests of mitochondrial DNA and of recombinant plasmids carrying specific restriction fragments, we have located the tRNA genes on the mitochondrial DNA. We have found that the mitochondrial tRNA genes are present in two major clusters, one between the two ribosomal RNA genes and the second closely following the large rRNA gene. Only one of the two DNA strands within these clusters codes for tRNAs. All of the genes for the seven specific purified tRNAs examined--those for alanine, formylmethionine, leucine 1, leucine 2, threonine, tyrosine, and valine--lie within these clusters. Interestingly, the formylmethionine tRNA hybridizes to two loci within one of these gene clusters. We have obtained a fairly detailed restriction map of part of this cluster and have shown that the two "putative" genes for formylmethionine tRNA are not arranged in tandem but are separated by more than 900 base pairs and by at least two other tRNA genes, those for alanine and for leucine 1 tRNAs.     

Mitochondrial and ribosomal DNA variation among members of the Anopheles quadrimaculatus (Diptera: Culicidae) species complex.

The extent of intra- and inter-specific variation in mitochondrial DNA and nuclear ribosomal RNA gene restriction sites was determined for the four sibling species of the Anopheles quadrimaculatus complex. Individual mosquitoes were identified by allozyme analysis according to previously published keys, and the total genomic DNA of these same individuals was then cleaved with restriction enzymes. Restriction maps of mitochondrial DNA, including the positions of variable sites, were constructed for each species. No evidence for interspecific hybridization was found in the populations surveyed. There was little variation in restriction patterns within any given species, but differences occurred among the four. Three restriction enzymes (AvaI, HindIII, and PvuII) yielded species-specific DNA restriction patterns for the mitochondrial DNA, while AvaI and HindIII produced diagnostic patterns for the ribosomal DNA. Thus, restriction patterns were very useful for detecting cryptic species but less appropriate than isozymes for studying genetic structure of populations within species.     

Rearrangements in the mitochondrial genome of somatic hybrid cell lines of Pennisetum americanum (L.) K. Schum. + Panicum maximum Jacq.

Summary Mitochondrial DNA from three somatic hybrid cell lines of Pennisetum americanum + Panicum maximum was compared with mitochondrial DNA of the parents. Gel electrophoresis of BamHI-restricted mitochondrial DNA indicated that extensive rearrangements had occurred in each of the three hybrid lines. The hybrid restriction patterns showed a combination of some bands from each parent plus novel fragments not present in either parent. Additional evidence for rearrangements was obtained by hybridization of eight DNA probes, carrying sequences of known coding regions, to Southern blots. Each of the somatic hybrids exhibited a partial combination of the parental mitochondrial genomes. These data suggest recombination or amplification of the mitochondrial genomes in the somatic hybrids.     

Effect of methionine dietary supplementation on mitochondrial oxygen radical generation and oxidative DNA damage in rat liver and heart

Methionine restriction without energy restriction increases, like caloric restriction, maximum longevity in rodents. Previous studies have shown that methionine restriction strongly decreases mitochondrial reactive oxygen species (ROS) production and oxidative damage to mitochondrial DNA, lowers membrane unsaturation, and decreases five different markers of protein oxidation in rat heart and liver mitochondria. It is unknown whether methionine supplementation in the diet can induce opposite changes, which is also interesting because excessive dietary methionine is hepatotoxic and induces cardiovascular alterations. Because the detailed mechanisms of methionine-related hepatotoxicity and cardiovascular toxicity are poorly understood and today many Western human populations consume levels of dietary protein (and thus, methionine) 2–3.3 fold higher than the average adult requirement, in the present experiment we analyze the effect of a methionine supplemented diet on mitochondrial ROS production and oxidative damage in the rat liver and heart mitochondria. In this investigation male Wistar rats were fed either a L-methionine-supplemented (2.5 g/100 g) diet without changing any other dietary components or a control (0.86 g/100 g) diet for 7 weeks. It was found that methionine supplementation increased mitochondrial ROS generation and percent free radical leak in rat liver mitochondria but not in rat heart. In agreement with these data oxidative damage to mitochondrial DNA increased only in rat liver, but no changes were observed in five different markers of protein oxidation in both organs. The content of mitochondrial respiratory chain complexes and AIF (apoptosis inducing factor) did not change after the dietary supplementation while fatty acid unsaturation decreased. Methionine, S-AdenosylMethionine and S-AdenosylHomocysteine concentration increased in both organs in the supplemented group. These results show that methionine supplementation in the diet specifically increases mitochondrial ROS production and mitochondrial DNA oxidative damage in rat liver mitochondria offering a plausible mechanism for its hepatotoxicity.     

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.