Novel features of metazoan mtDNA revealed from sequence analysis of three mitochondrial DNA segments of the land snail Albinaria turrita (Gastropoda: Clausiliidae)

The mitochondrial DNA (mtDNA) size of the terrestrial gastropod Albinaria turrita was determined by restriction enzyme mapping and found to be approximately 14.5 kb. Its partial gene content and organization were examined by sequencing three cloned segments representing about one-fourth of the mtDNA molecule. Complete sequences of cytochrome c oxidase subunit II (COII), and ATPase subunit 8 (ATPase8), as well as partial sequences of cytochrome c oxidase subunit I (COI), NADH dehydrogenase subunit 6 (ND6), and the large ribosomal RNA (IrRNA) genes were determined. Nine putative tRNA genes were also identified by their ability to conform to typical mitochondrial tRNA secondary structures. An 82-nt sequence resembles a noncoding region of the bivalve Mytilus edulis, even though it might contain a tenth tRNA gene with an unusual 5-nt overlap with another tRNA gene. The genetic code of Albinaria turrita appears to be the same as that of Drosophila and Mytilus edulis. The structures of COI and COII are conservative, but those of ATPase8 and ND6 are diversified. The sequenced portion of thelrRNA gene (1,079 nt) is characterized by conspicuous deletions in the 5 and 3 ends; this gene represents the smallest coelomate IrRNA gene so far known. Sequence comparisons of the identified genes indicate that there is greater difference between Albinaria and Mytilus than between Albinaria and Drosophila. An evolutionary analysis, based on COII sequences, suggests a possible nonmonophyletic origin of molluskan mtDNA. This is supported also by the absence of the ATPase8 gene in the mtDNA of Mytilus and nematodes, while this gene is present in the mtDNA of Albinaria and Cepaea nemoralis and in all other known coelomate metazoan mtDNAs.     

Relative Rates of Nucleotide Substitution in Frogs

Accurate estimation of relative mutation rates of mitochondrial DNA (mtDNA) and single-copy nuclear DNA (scnDNA) within lineages contributes to a general understanding of molecular evolutionary processes and facilitates making demographic inferences from population genetic data. The rate of divergence at synonymous sites (K_s) may be used as a surrogate for mutation rate. Such data are available for few organisms and no amphibians. Relative to mammals and birds, amphibian mtDNA is thought to evolve slowly, and the K_s ratio of mtDNA to scnDNA would be expected to be low as well. Relative K_s was estimated from a mitochondrial gene, ND2, and a nuclear gene, c-myc, using both approximate and likelihood methods. Three lineages of congeneric frogs were studied and this ratio was found to be approximately 16, the highest of previously reported ratios. No evidence of a low K_s in the nuclear gene was found: c-myc codon usage was not biased, the K_s was double the intron divergence rate, and the absolute K_s was similar to estimates obtained here for other genes from other frog species. A high K_s in mitochondrial vs. nuclear genes was unexpected in light of previous reports of a slow rate of mtDNA evolution in amphibians. These results highlight the need for further investigation of the effects of life history on mutation rates. Current address (Andrew J. Crawford): Smithsonian Tropical Research Institute, Apartado 2072, Balboa, Ancon, Republic of Panama     

Behavior of mitochondria and their nuclei during amoebae cell proliferation inPhysarum polycephalum

Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system - BrdU 5-bromodeoxyuridine - FITC fluorescein isothiocyanate     

Drosophila mitochondrial DNA: Conserved sequences in the A+T-rich region and supporting evidence for a secondary structure model of the small ribosomal RNA

Summary The sequence of a segment of theDrosophila virilis mitochondrial DNA (mtDNA) molecule that contains the A+T-rich region, the small rRNA gene, the tRNA^f-met, tRNA^gln, and tRNA^ile genes, and portions of the ND2 and tRNA^val genes is presented and compared with the corresponding segment of theD. yakuba mtDNA molecule. The A+T-rich regions ofD. virilis andD. yakuba contain two correspondingly located sequences of 49 and 276/274 nucleotides that appear to have been conserved during evolution. In each species the replication origin of the mtDNA molecule is calculated to lie within a region that overlaps the larger conserved sequence, and within this overlap is found a potential hairpin structure. Substitutions between the larger conserved sequences of the A+T-rich regions, the small mt-rRNA genes, and the ND2 genes are biased in favor of transversions, 71–97% of which are AT changes. There is a 13.8 times higher frequency of nucleotide differences between the 5 halves than between the 3 halves of theD. virilis andD. yakuba small mt-rRNA genes. Considerations of the effects of observed substitutions and deletion/insertions on possible nucleotide pairing within the small mt-rRNA genes ofD. virilis andD. yakuba strongly support the secondary structure model for theDrosophila small mt-rRNA that we previously proposed.     

The use of mitochondrial DNA polymorphism in the classification of individual onion plants by cytoplasmic genotypes

Summary Individual plants of a Japanese onion variety Sapporo-ki, which is characterized by the occasional occurrence of male-sterile plants, have been investigated for mitochondrial (mt) DNA polymorphism. Male-fertile and the Jones' cytoplasmic male-sterile (CMS) onions were also included for comparison. Southern blot hybridization with rrn26, cox-I, cox-II, cob, atpA and atp9 genes as probes revealed the two classes of mtDNA variation within a population of Sapporo-ki: Out of the 41 plants examined 19 contained mtDNA typical of malefertile plants, and 22 individuals contained mtDNA typical of the Jones' CMS genotype. Our results thus indcate that the use of the mitochondrial gene probes may greatly facilitate the classification of individual plants by cytoplasmic genotypes.     

In vivo homologous recombination intermediates of yeast mitochondrial DNA analyzed by electron microscopy

Summary To study the structure of in vivo mitochondrial DNA recombination intermediates in Saccharomyces cerevisiae, we used a deletion mutant of the wild type mitochondrial genome. The mtDNA of this petite is composed of a direct tandem repetition of an 4,600 pb monomer repeat unit with a unique HhaI restriction enzyme site per repeat. The structure of native mtDNA isolated from log phase cells, and mtDNA crosslinked in vivo with trioxsalen plus UVA irradiation, was studied by electron microscopy. Both populations contained crossed strand Holliday type recombination intermediates. Digestion of both non-crosslinked and crosslinked and mtDNA with the enzyme HhaI released X and H shaped structures composed of two monomers. Electron microscopic analysis revealed that these structures had pairs of equal length arms as required for homologous recombination intermediates and that junctions could occur at points along the entire monomer length. The percentage of recombining monomers in both non-crosslinked and trioxsalen crosslinked mtDNA was calculated by quantitative analysis of all the structures present in an HhaI digest. The relationship between these values and the apparent dispersive replication of mtDNA in density-shift experiments and mtDNA fragility during isolation is discussed.     

Mitochondrial DNA of the sea anemone,Metridium senile (Cnidaria): Prokaryote-like genes for tRNAf-Met and small-subunit ribosomal RNA,and standard genetic code specificities for AGR and ATA codons

The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the sea anemone Metridium senile (phylum Cnidaria, class Anthozoa, order Actiniaria) has been determined, within which have been identified the genes for respiratory chain NADH dehydrogenase subunit 2 (ND2), the small-subunit rRNA (s-rRNA), cytochrome c oxidase subunit II(COII), ND4, ND6, cytochrome b (Cyt b), tRNA^f-Met, and the large-subunit rRNA (1-rRNA). The eight genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of the M. senile mt-genes differs from that of other metazoan mtDNAs. In M. senile mt-protein genes, AGA and AGG codons appear to have the standard genetic code specification of arginine, rather than serine as found for other invertebrate mt-genetic codes. Also, ATA has the standard genetic code specification of isoleucine. TGA occurs in three M. senile mt-protein genes and may specify tryptophan as in other metazoan, protozoan, and some fungal mt-genetic codes. The M. senile mt-rRNA^f-Met gene has primary and secondary structure features closely resembling those of the Escherichia coli initiator tRNA, including standard dihydrouridine and TC loop sequences and a mismatch pair at the top of the aminoacyl stem. Determinations of the 5 and 3 end nucleotides of the M. senile mt-srRNAs indicated that these molecules have a homogenous size of 1,081 ntp, larger than any other known metazoan mt-s-rRNAs. Consistent with its larger size, the M. senile mt-s-rRNA can be folded into a secondary structure that more closely resembles that of the E. coli 16S rRNA than can any other metazoan mt-s-rRNA. These findings concerning M. senile mtDNA indicate that most of the unusual features regarding metazoan mt-genetic codes, rRNAs, and probably tRNAs developed after divergence of the Cnidarian line from the ancestral line common to other metazoa.Correspondence to: D.R. Wolstenholme     

Platyhelminth mitochondrial DNA: Evidence for early evolutionary origin of a tRNAserAGN that contains a dihydrouridine arm replacement loop,and of serine-specifying AGA and AGG codons

Summary The nucleotide sequence of a segment of the mitochondrial DNA (mtDNA) molecule of the liver flukeFasciola hepatica (phylum Platyhelminthes, class Trematoda) has been determined, within which have been identified the genes for tRNA^ala, tRNA^asp, respiratory chain NADH dehydrogenase subunit I (ND1), tRNA^asn, tRNA^pro, tRNA^ile, tRNA^lys, ND3, tRNA^serAGN, tRNA^trp, and cytochromec oxidase subunit I (COI). The 11 genes are arranged in the order given and are all transcribed from the same strand of the molecule. The overall order of theF. hepatica mitochondrial genes differs from what is found in other metazoan mtDNAs. All of the sequenced tRNA genes except the one for tRNA^serAGN can be folded into a secondary structure with four arms resembling most other metazoan mitochondrial tRNAs, rather than the tRNAs that contain a TψC arm replacement loop, found in nematode mtDNAs. TheF. hepatica mitochondrial tRNA^serAGN gene contains a dihydrouridine arm replacement loop, as is the case in all other metazoan mtDNAs examined to date. AGA and AGG are found in theF. hepatica mitochondrial protein genes and both codons appear to specify serine. These findings concerningF. hepatica mtDNA indicate that both a dihydrouridine arm replacement loop-containing tRNA^serAGN gene and the use of AGA and AGG codons to specify serine must first have occurred very early in, or before, the evolution of metazoa.     

The complete nucleotide sequence and multipartite organization of the tobacco mitochondrial genome: comparative analysis of mitochondrial genomes in higher plants

Tobacco is a valuable model system for investigating the origin of mitochondrial DNA (mtDNA) in amphidiploid plants and studying the genetic interaction between mitochondria and chloroplasts in the various functions of the plant cell. As a first step, we have determined the complete mtDNA sequence of Nicotiana tabacum. The mtDNA of N. tabacum can be assumed to be a master circle (MC) of 430,597 bp. Sequence comparison of a large number of clones revealed that there are four classes of boundaries derived from homologous recombination, which leads to a multipartite organization with two MCs and six subgenomic circles. The mtDNA of N. tabacum contains 36 protein-coding genes, three ribosomal RNA genes and 21 tRNA genes. Among the first class, we identified the genes rps1 and rps14, which had previously been thought to be absent in tobacco mtDNA on the basis of Southern analysis. Tobacco mtDNA was compared with those of Arabidopsis thaliana, Beta vulgaris, Oryza sativa and Brassica napus. Since repeated sequences show no homology to each other among the five angiosperms, it can be supposed that these were independently acquired by each species during the evolution of angiosperms. The gene order and the sequences of intergenic spacers in mtDNA also differ widely among the five angiosperms, indicating multiple reorganizations of genome structure during the evolution of higher plants. Among the conserved genes, the same potential conserved nonanucleotide-motif-type promoter could only be postulated for rrn18-rrn5 in four of the dicotyledonous plants, suggesting that a coding sequence does not necessarily move with the promoter upon reorganization of the mitochondrial genome.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

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.     

DNA synthesis in isolated mitochondrial nucleoids from plasmodia ofPhysarum polycephalum

Summary A mitochondrion contains multiple copies of mitochondrial DNA (mtDNA) in the mitochondrial nucleoid (mt-nucleoid, synonym for mitochondrial nuclei). Replicaton of mtDNA in the mtnucleoids appears to be regulated within groups of adjacent mtDNA molecules, known as mitochondrial replicon clusters (MRCs). In this study, we isolated structurally intact mt-nucleoids from the plasmodia ofPhysarum polycephalum and characterized DNA synthesis in the isolated mt-nucleoids. The mt-nucleoids were isolated by dissolving the membranes of highly purified mitochondria with 0.5% Nonidet P-40. The structural integrity of the isolated mt-nucleoid was determined by observing the rod shape of the mt-nucleoid and the structure of the MRC. The isolated mt-nucleoids required four deoxyribonucleoside triphosphates and MgCl₂ for DNA synthesis. The DNA synthesis was resistant to aphidicolin and showed only low sensitivity to N-ethylmaleimide and to ddTTP, suggesting that the DNA synthesis is catalyzed by plant-type mitochondrial DNA polymerase. The capacity for DNA synthesis in the isolated mt-nucleoids was similar to that in the isolated mitochondria, despite removal of most of the mitochondrial matrix and membrane. Furthermore, visualization of sites of DNA synthesis in vitro revealed that DNA synthesis in the isolated mt-nucleoids occurred in each MRC. These results suggest that the isolated mt-nucleoids are capable of efficient and systematic DNA synthesis in vitro. Therefore, the use of isolated mt-nucleoids should permit in vitro characterization of the molecular mechanism of mtDNA replication in the MRC.Abbreviations BrdU 5-bromodeoxyuridine - BrdUTP 5-bromo-deoxyuridine triphosphate - DAPI 4,6-diamidino-2-phenylindole - dNTP deoxyribonucleoside triphosphate - ddCTP dideoxycytidine triphosphate - NEM N-ethylmaleimide - MRC mitochondrial replicon cluster; mt mitochondrial - NP-40 Nonidet P-40 - PBS phosphatebuffered saline - PMSF phenylmethanesulfonyl fluoride - rNTP ribonucleoside triphosphate - VIMPCS video-intensified microscope photon-counting system     

Nuclear-mitochondrial interactions in cytoplasmic male-sterile sorghum

Summary Variation in mitochondrial genome organization and expression between male fertile and sterile nuclear-cytoplasmic combinations of sorghum has been examined. Cytoplasmic genotypes were classified into eleven groups on the basis of restriction endonuclease digestion of mitochondrial DNA (mtDNA) and five groups on the basis of mitochondrial translation products. These cytoplasms were further characterized by hybridization of specific gene probes to Southern blots of EcoRI digested mtDNA, and identification of the fragment location of four mitochondrial genes. Variation was observed in the genomic location and copy number of the F₁ ATPase -subunit gene, as well as the genomic location and gene product of the cytochrome c oxidase subunit I gene. The effect of nuclear genotype on mitochondrial genome organization, expression and the presence of two linear plasmid-like mtDNA molecules was examined. Our results indicate that nuclear-mitochondrial interactions are required for regulation of mitochondrial gene expression. When a cytoplasm is transferred from its natural to a foreign nuclear background some changes in the products of in organello mitochondrial protein synthesis occur. In a number of cytoplasmic genotypes these changes correlate with the expression of cytoplasmic male sterile phenotype, suggesting a possible molecular basis for this mutation.     

Rearrangement,amplification, and assortment of mitochondrial DNA molecules in cultured cells of Brassica campestris

Summary We compared Brassica campestris mitochondrial and chloroplast DNAs from whole plants and from a 2-year-old cell culture. No differences were observed in the chloroplast DNAs (cpDNAs), whereas the culture mitochondrial DNA (mtDNA) was extensively altered. Hybridization analysis revealed that the alterations are due entirely to rearrangement. At least two inversions and one large duplication are found in the culture mtDNA. The duplication element is shown to have the usual properties of a plant mtDNA high frequency recombination repeat. The culture mtDNA exists as a complex heterogeneous population of rearranged and unrearranged molecules. Some of the culture-associated rearranged molecules are present in low levels in native plant tissue and appear to have sorted out and amplified in the culture. Other mtDNA rearrangements may have occurred de novo. In addition to alterations of the main mitochondrial genome, an 11.3 kb linear mtDNA plasmid present in whole plants is absent from the culture. Contrary to findings in cultured cells of other plants, small circular mtDNA molecules were not detected in the B. campestris cell culture.     

The Mitochondrial Sequences of Heptathela hangzhouensis and Ornithoctonus huwena Reveal Unique Gene Arrangements and Atypical tRNAs

We have sequenced the complete mitochondrial genomes of the spiders Heptathela hangzhouensis and Ornithoctonus huwena. Both genomes encode 13 protein-coding genes, 22 tRNA genes, and 2 ribosomal RNA genes. H. hangzhouensis, a species of the suborder Mesothelae and a representative of the most basal clade of Araneae, possesses a gene order identical to that of Limulus polyphemus of Xiphosura. On the other hand, O. huwena, a representative of suborder Opisthothelae, infraorder Mygalomorphae, was found to have seven tRNA genes positioned differently from those of Limulus. The rrnL–trnL1–nad1 arrangement shared by the araneomorph families Salticidae, Nesticidae, and Linyphiidae and the mygalomorph family Theraphosidae is a putative synapomorphy joining the mygalomorph with the araneomorph. Between the two species examined, base compositions also differ significantly. The lengths of most protein-coding genes in H. hangzhouensis and O. huwena mtDNA are either identical to or slightly shorter than their Limulus counterparts. Usage of initiation and termination codons in these protein-coding genes seems to follow patterns conserved among most arthropod and some other metazoan mitochondrial genomes. The sequences of the 3 ends of rrnS and rrnL in the two species are similar to those reported for Limulus, and the entire genes are shortened by about 100–250 nucleotides with respect to Limulus. The lengths of most tRNA genes from the two species are distinctly shorter than those of Limulus and the sequences reveal unusual inferred tRNA secondary structures. Our finding provides new molecular evidence supporting that the suborder Mesothelae is basal to opisthothelids.Reviewing Editor Dr. Rafael Zardoya     

The cytochrome oxidase II gene in mitochondria of the sugar-beetBeta vulgaris L.

We have cloned and analyzed the sugar-beet mitochondrial gene for cytochrome oxidase subunit II (coxII). The sugar-beet and its deduced amino acid sequence were compared to its homologouscoxII gene sequences from both monocot and dicot plants. It was found to be highly conserved (89–95%) compared to homologue in other plant species. The 780 bp coding sequence of the sugar beetcoxII gene is interrupted at position 383 by a 1463 bp intron. This intron contains an additional 107 bp sequence that is not found in any of the plantcoxII genes studied thus far. The structure of the intron suggests that a large intron existed in an ancestralcoxII gene before monocots and dicots diverged in evolution. Three CGG codons in the sugar-beetcoxII coding sequence align with conserved tryptophan residues in the homologous gene of other species, suggesting that RNA editing takes place also in sugar-beet mitochondria. In 13 out of 24 codons ofcoxII mRNA that were found to be edited in four other plants, the sugar-beet gene already utilizes the edited codons. This phenomenon may indicate that the mitochondrial genome in sugar-beet is phylogenetically more archaic relative to these plants. An additional sequence of 279 bp that is identical to the first exon ofcoxII was identified in the mtDNA of the sugar-beet. This pseudo-gene is transcribed and its existence in the mitochondrial genome is unexplained.     

A vital function for mitochondrial DNA in the petite-negative yeastKluyveromyces lactis

Petite-negative yeasts do not form viable respiratory-deficient mutants on treatment with DNA-targeting drugs that readily eliminate the mitochondial DNA (mtDNA) from petite-positive yeasts. However, in the petite-negative yeastKluyveromyces lactis, specific mutations in the nuclear genesMGI2 andMGI5 encoding the- and-subunits of the mitochondrial F₁-ATPase, allow mtDNA to be lost. In this study we show that wild-typeK. lactis does not survive in the absence of its mitochondrial genome and that the function ofmgi mutations is to suppress lethality caused by loss of mtDNA. Firstly, we find that loss of a multicopy plasmid bearing amgi allele readily occurs from a wild-type strain with functional mtDNA but is not tolerated in the absence of mtDNA. Secondly, we cloned theK. lactis homologue of theSaccharomyces cerevisiae mitochondrial genome maintenance geneMGM101, and disrupted one of the two copies in a diploid. Following sporulation, we find that segregants containing the disrupted gene form minicolonies containing 6-8000 inviable cells. By contrast, disruption ofMGM101 is not lethal in a haploidmgi strain with a specific mutation in a subunit of the mitochondrial F₁-ATPase. These observations suggest that mtDNA inK. lactis encodes a vital function which may reside in one of the three mitochondrially encoded subunits of F₀.     

Organization of heterogeneous mitochondrial DNA molecules in mitochondrial nuclei of cultured tobacco cells

Summary The molecular size of mitochondrial DNA (mtDNA) molecules and the number of copies of mtDNA per mitochondrion were evaluated from cultured cells of the tobacco BY-2 line derived fromNicotiana tabacum L. cv. Bright Yellow-2. To determine the DNA content per mitochondrion, protoplasts of cultured cells were stained with 4,6-diamidino-2-phenylindole (DAPI), and the intensity of the fluorescence emitted from the mitochondrial nuclei (mt-nuclei) was measured with a video-intensified photon counting microscope system (VIM system). Each mitochondrion except for those undergoing a division contained one mt-nucleus. The most frequently measured size of the DNA in the mitochondria was between 120 and 200 kilobase pairs (kbp) throughout the course of culture of the tobacco cells. Mitochondria containing more than 200 kbp of DNA increased significantly in number 24 h after transfer of the cells into fresh medium but their number fell as the culture continued. Because division of mitochondria began soon after transfer of the cells into fresh medium and continued for 3 days, the change of the DNA content per mitochondrion during the culture must correspond to DNA synthesis of mitochondria in the course of mitochondrial division. By contrast, the analyses of products of digestion by restriction endonucleases indicated that the genome size of the mtDNA was at least 270 kbp. Electron microscopy revealed that mtDNAs were circular molecules and their length ranged from 1 to 35 m, and 60% of them ranged from 7 to 11 rn. These results indicate that the mitochondrial genome in tobacco cells consists of multiple species of mtDNA molecules, and mitochondria do not contain all the mtDNA species. Therefore, mitochondria are heterogeneous in mtDNA composition.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - mtDNA mitochondrial DNA - mt-genome mitochondrial genome - mt-nucleus mitochondrial nucleus - ptDNA proplastid DNA - pt-nucleus proplastid nucleus - VIM system video-intensified photon counting microscope system     

Nucleotide Content Gradients in Maternally and Paternally Inherited Mitochondrial Genomes of the Mussel <Emphasis Type="Italic">Mytilus</Emphasis>

Several studies have shown that in vertebrate mtDNAs the nucleotide content at fourfold degenerate sites is well correlated with the site’s time of exposure to the single-strand state, as predicted from the asymmetrical model of mtDNA replication. Here we examine whether the same explanation may hold for the regional variation in nucleotide content in the maternal and paternal mtDNAs of the mussel Mytilus galloprovincialis. The origin of replication of the heavy strand (O_H) of these genomes has been previously established. A systematic search of the two genomes for sequences that are likely to act as the origin of replication of the light strand (O_L) suggested that the most probable site lies within the ND3 gene. By adopting this O_L position we calculated times of exposure for 0_FD (nondegenerate), 2_FD (twofold degenerate), and 4_FD (fourfold degenerate) sites of the protein-coding part of the genome and for the rRNA, tRNA and noncoding parts. The presence of thymine and absence of guanine at 4_FD sites was highly correlated with the presumed time of exposure. Such an effect was not found for the 2_FD sites, the rRNA, the tRNA, or the noncoding parts. There was a trend for a small increase in cytosine at 0_FD sites with exposure time, which is explicable as the result of biased usage of 4_FD codons. The same analysis was applied to a recently sequenced mitochondrial genome of Mytilus trossulus and produced similar results. These results are consistent with the asymmetrical model of replication and suggest that guanine oxidation due to single-strand exposure is the main cause of regional variation of nucleotide content in Mytilus mitochondrial genomes. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users. [Reviewing Editor: Dr. David Pollock]     

Comparison between the complete mtDNA sequences of the blue and the fin whale,two species that can hybridize in nature

The sequence of the mitochondrial DNA (mtDNA) molecule of the blue whale (Balaenoptera musculus) was determined. The molecule is 16,402 by long and its organization conforms with that of other eutherian mammals. The molecule was compared with the mtDNA of the congeneric fin whale (B. physalus). It was recently documented that the two species can hybridize and that male offspring are infertile whereas female offspring may be fertile. The present comparison made it possible to determine the degree of mtDNA difference that occurs between two species that are not completely separated by hybridization incompatibility. The difference between the complete mtDNA sequences was 7.4%. Lengths of peptide coding genes were the same in both species. Except for a small portion of the control region, disruption in alignment was usually limited to insertion/deletion of a single nucleotide. Nucleotide differences between peptide coding genes ranged from 7.1 to 10.5%, and difference at the inferred amino acid level was 0.0–7.9%. In the rRNA genes the mean transition difference was 3.8%. This figure is similar in degree to the difference (3.4%) between the 12S rRNA gene of humans and the chimpanzee. The mtDNA differences between the two whale species, involving both peptide coding and rRNA genes, suggest an evolutionary separation of 5 million years. Although hybridization between more distantly related mammalian species may not be excluded, it is probable that the blue and fin whales are nearly as different in their mtDNA sequences as hybridizing mammal species may be. Correspondence to: Ú. Árnason     

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea starPisaster ochraceus

Summary We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea starPisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA^glu and tRNA^thr are 3 to the 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.