Organisation of the mitochondrial genome from Atlantic salmon (Salmo salar)

A restriction map of Atlantic salmon mitochondrial DNA was constructed. The smallest XbaI fragment of the salmon mitochondrial genome was cloned and subjected to partial DNA sequence analysis. This fragment contains the genes for ATPase 6 and cytochrome oxidase III. The putative organisation of the mitochondrial genome relative to the physical map is shown.     

Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages.

The mitochondrial DNA (mtDNA) sequence variation of the South American Ticuna, the Central American Maya, and the North American Pima was analyzed by restriction-endonuclease digestion and oligonucleotide hybridization. The analysis revealed that Amerindian populations have high frequencies of mtDNAs containing the rare Asian RFLP HincII morph 6, a rare HaeIII site gain, and a unique AluI site gain. In addition, the Asian-specific deletion between the cytochrome c oxidase subunit II (COII) and tRNA(Lys) genes was also prevalent in both the Pima and the Maya. These data suggest that Amerindian mtDNAs derived from at least four primary maternal lineages, that new tribal-specific variants accumulated as these mtDNAs became distributed throughout the Americas, and that some genetic variation may have been lost when the progenitors of the Ticuna separated from the North and Central American populations.     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis. Denaturation mapping by electron microscopy.

Electronmicroscopic observation of the denaturation pattern of 130 partially denaturated linear mitochondrial DNA molecules from Saccharomyces carlsbergensis was used to investigate the distribution of AT-rich sequences within the mitochondrial genome. The molecules were observed after heating to 43 degrees C in the presence of 12% formaldehyde. These conditions resulted in an average denaturation per molecule of 21%. The average length of the molecules was 10 mum, and a few molecules had a length corresponding to the size of the complete genome. The undenaturated regions varied in length from 0.1 to 5.0 mum with denaturated regions of length 0.02 to 0.1 mum in between. A denaturation map was constructed by use of one of the long molecules (28.7 mum) as a master molecule for positioning of all other molecules. This map shows distinct regions corresponding to the position of easily denaturated sequences in the mitochondrial DNA. These sequences which presumably correspond to the very AT-rich regions, known to exist in the yeast mitochondrial DNA, were found at intervals of about 0.5 - 3 mum on the map.     

Mitochondrial genome structure of rice suspension culture from cytoplasmic male-sterile line (A-58CMS): reappraisal of the master circle

Summary The mitochondrial DNA (mtDNA) from the cultured cells of a cytoplasmic male-sterile line (A-58CMS) of rice (Oryza sativa) was cloned and its physical map was constructed. There was structural alteration on the mitochondrial genome during the cell culture. Detailed restriction analysis of cosmid clones having mtDNA fragments suggested either that the master genome has a 100-kb duplication (the genome size becomes 450 kb) or that a master circle is not present in the genome (the net structural complexity becomes 350 kb). The physical map of plant mitochondrial genomes thus far reported is illustrated in a single circle, namely a master circle. However, no circular DNA molecule corresponding to a master circle has yet been proved. In the present report, representation of plant mitochondrial genomes and a possibility for mitochondrial genome without a master circle are discussed.     

A restriction endonuclease cleavage map of mouse mitochondrial DNA.

A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized PstI and four sites recognized by BamI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by KpnI or SalI. The migration of linear DNA with a molecular weight greater than 1 X 10(6) was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^? mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

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.     

Organization of tRNA and rRNA genes in N. crassa mitochondria: intervening sequence in the large rRNA gene and strand distribution of the RNA genes.

Through analysis of cloned fragments of N. crassa mitochondrial DNA, we have derived a physical map for the region of the mitochondrial genome which encodes the ribosomal RNAs and most of the tRNAs. We have located RNA genes on this map by hybridization of purified 32P end-labeled RNA probes, and our findings are as follows. First, the gene for the large ribosomal RNA contains an intervening sequence of approximately 2000 bp. Second, the genes for the small and large ribosomal RNAs are not adjacent, as previously reported, and the region between them contains a number of tRNA genes, including that for the mitochondrial tRNATyr, which is located close to the small rRNA gene on the same strand of the mitochondrial DNA. Third, there is a second cluster of tRNA genes on the mitochondrial DNA following the large ribosomal RNA gene, but there is no evidence for the presence of tRNA genes in the intervening sequence of the large ribosomal RNA. Fourth, hybridization of labeled ribosomal and transfer RNAs to the separated strands of a cloned 16 kbp DNA fragment covering this region indicates that the two ribosomal RNAs and most, if not all, of the mitochondrial tRNAs are encoded on one strand of the mitochondrial DNA.     

Bidirectional replication initiates at sites throughout the mitochondrial genome of birds

Analysis of mitochondrial replication intermediates of Gallus gallus on fork-direction gels indicates that replication occurs in both directions around circular mitochondrial DNA. This finding was corroborated by a study of chick mitochondrial DNA on standard neutral two-dimensional agarose gels, which yielded archetypal initiation arcs in fragments covering the entire genome. There was, however, considerable variation in initiation arc intensity. The majority of initiation events map to regions flanking the major non-coding region, in particular the NADH dehydrogenase subunit 6 (ND6) gene. Initiation point mapping of the ND6 gene identified prominent free 5' ends of DNA, which are candidate start sites for DNA synthesis. Therefore we propose that the initiation zone of G. gallus mitochondrial DNA encompasses most, if not all, of the genome, with preferred initiation sites in regions flanking the major non-coding region. Comparison with mammals suggests a common mechanism of initiation of mitochondrial DNA replication in higher vertebrates.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

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.     

Taenia hydatigena: isolation of mitochondrial DNA, molecular cloning, and physical mitochondrial genome mapping

Mitochondrial DNA was isolated from Taenia hydatigena, T. crassiceps, and Echinococcus granulosus using a cetyltrimethylammonium bromide precipitation technique. The technique is simple, rapid, reproducible, and does not require extensive high speed ultracentrifugation. The advantage of using mitochondrial DNA from taeniid cestodes for comparative restriction analysis was demonstrated. Mitochondrial DNA of T. hydatigena was isolated as covalently closed circular molecules. These were linearized by single digestion with BamHI and the molecular weight was estimated from the linear form of 17.6 kb. The mitochondrial DNA of T. hydatigena is therefore similar in size and structure to that of many other animal species. The entire mitochondrial genome was cloned into pBR322 in Escherichia coli and a restriction map of the recombinant molecule was constructed. The potential of using the cloned mitochondrial genome as a probe in speciation studies as well as for providing functional information on the role of the cestode mitochondrion is discussed.     

The sugar beet mitochondrial genome: A complex organisation generated by homologous recombination

Summary The complete physical map of the mitochondrial genome of the Owen cytoplasm of sugar beet has been determined from overlapping cosmid clones. The genome is 386 kb in size and has a multicircular organisation generated by homologous recombination across repeated DNA elements. The location of the rRNA genes and several polypeptide genes has been determined. In addition the mitochondrial genome was found to contain a sequence of chloroplast DNA including part of the 16 S rRNA gene.     

Identification of three populations ofOphiostoma ulmi (aggressive subgroup) by mitochondrial DNA restriction-site mapping and nuclear DNA fingerprinting

Three genetically distinct populations of the Dutch elm pathogenOphistoma ulmi within the aggressive subgroup were defined by the hybridization of a human minisatellite DNA sequence (HVR 33.6) to polymorphic dispersed DNA sequences within theO. ulmi nuclear genomes. For the 10 isolates examined there was a close correlation between nuclear DNA fingerprints and mitochondrial (mt) DNA restriction patterns. A restriction-site map was constructed for the mitochondrial genomes for each of these populations. The three mt DNA maps corresponded to genome sizes of 49.1 (Type I), 49.9 (Type II), and 53.9 (Type III) kilobase pairs (kbp) of DNA. The Type I and Type II mt genomes differed from the Type III mt genome by discrete length mutations of 4.8 and 4.0 kbp, respectively. It is unknown whether these length mutations resulted from insertions into or deletions from a progenitor mitochondrial genome. There was no correlation between the mitochondrial or nuclear genotypes and the geographical source of the isolates.     

Denaturation map of the circular mitochondrial genome of Neurospora crassa.

A denaturation map of mitochondrial DNA from the wild type strain 5256 of Neurospora crassa was constructed by computer analysis of the contour length distribution of single- and double-stranded regions of nineteen circular and three full length linear molecules after partial denaturation. The data suggest that mitochondrial DNA in this strain is a homogeneous population of a circular molecule of molecular weight 41 - 10(6) with an asymmetric distribution of AT-rich regions, and that linear molecules derive from this genome by random breaks during isolation.     

A new estimate of sequence divergence of mitochondrial DNA using restriction endonuclease mappings

Summary A new estimate of the sequence divergence of mitochondrial DNA in related species using restriction enzyme maps is constructed. The estimate is derived assuming a simple Posisson-like model for the evolutionary process and is chosen to maximize an expression which is a reasonable approximation to the true likelihood of the restriction map data. Using this estimate, four sets of mitochondrial DNA data are analyzed and discussed.     

Genetic and physical maps and a clone bank of mitochondrial DNA from rice

Summary Mitochondrial DNA (mtDNA) was isolated from young green leaves of rice plants. DNA fragments were cloned into lambda DNA, and clones that hybridized to mitochondrial genes from other plants were selected. Distal restriction fragments of these clones were used as probes for the selection of overlapping clones. A genetic map was finally created from the library by walking along the genome. The mitochondrial genome consists of five basic circles, with each circle sharing homologous sequences with one or two other circles. A master circle was constructed from the results of recombination across repeated sequences, and its size was estimated to be 492 kb. A physical map and a bank of overlapping clones were also constructed.