Physical mapping of the mitochondrial genome of the cultivated mushroom Agaricus brunnescens (= A. bisporus)

Summary Mitochondrial (mt) DNA from the commercial mushroom Agaricus brunnescens Peck [= A. bisporus (Lange) Imbach] was purified by cesium chloride/bisbenzimide gradient centrifugation. A physical map of the mtDNA fragments produced by BamHI, EcoRl, and PvuII digestion was generated by filter hybridizations with radiolabelled BamHI mtDNA probes. The A. brunnescens mtDNA was a circular molecule 136 kilo-basepairs (kbp) in length and contained an inverted repeat between 4.6 and 9.2 kbp in size. Orientational isomers of the mitochondrial genome were not detected. The positions of six genes were located on the A. brunnescens mtDNA map by heterologous hybridization. No coding function has yet been ascribed to the inverted repeat. The large rRNA gene was located on the smaller single copy region. The genes for cytochrome b, cytochrome oxidase (subunit III), ATPase (subunits 8 and 6) and the small rRNA were located on different regions of the larger single copy region.     

Involvement of a large inverted repeated sequence

Summary Southern hybridization of the total DNA of Agrocybe aegerita with cloned mitochondrial (mt) probes revealed a sequence homology between two distant mitochondrial restriction fragments. From the mtDNA restriction map and the distribution of restriction sites on the cross-hybridizing mitochondrial fragments, two copies of a large inverted repeated sequence (IR) of 3 kbp were located on the mitochondrial genome. These IR sequences divided the 80 kbp mtDNA into two singlecopy regions of 24 kbp (SSC) and 50 kbp (LSC). For the first time in higher fungi, this IR sequence has been shown to be involved in an intramolecular homologous recombinational event. Such a rearrangement led to an inversion of the orientation of the two unique-copy regions, without any change in mtDNA complexity. The location of the recombinational event was compared with previously reported plant and fungal mitochondrial rearrangements and the potential role of the IR sequence was discussed.     

Mitochondrial DNA in the aquatic fungus Allomyces

Summary The mitochondrial DNA from seven species of the aquatic phycomycete Allomyces has been isolated and characterized by restriction enzyme analysis. Comparison of the mitochondrial DNA restriction enzyme fragmentation patterns showed pronounced differences not only among species but also among four isolates of A. arbuscula. The mitochondrial DNAs range in size from 39 kbp in A. neo-moniliformis to 56 kbp in A. macrogynus.A physical map of the mitochondrial DNA of Allomyces arbuscula strain Costa Rica 21 has been constructed. The genome is circular and has a size of 49.2 kbp. The genes coding for the small and large ribosomal RNAs, cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, and ATPase subunits 6 and 9 were localized in the mitochondria) DNA by heterologous hybridization with specific mitochondria) gene probes from Saccaromyces cerevisiae and Neurospora crassa. Comparison of the gene map of the closely related species Blastocladiella emersonii with that of A. arbuscula indicates a similar gene order in the two organisms.     

Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3

Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.     

Mitochondrial DNA of the filamentous ascomycete Cochliobolus heterostrophus

Summary The mitochondrial chromosome of Cochliobolus heterostrophus is a circle approximately 115 kb in circumference, among the largest known from fungi. A physical map of C. heterostrophus mtDNA was constructed using the restriction enzymes BamHI, EcoRI, and PvulI by DNA-DNA hybridizations with cloned or purified mtDNA BamHI fragments. The following sequences were located on the mtDNA map: (1) the large and small mitochondrial ribosomal RNA genes (identified by heterologous hybridization to cloned Neurospora crassa rRNA genes); (2) the sequence homologous to a mitochondrial plasmid present in one field isolate of C. heterostrophus; and (3) a 1.05 kb EcoRI fragment that functions as an autonomously replicating sequence in Saccharomyces cerevisiae. An examination of mtDNA from 23 isolates of C. heterostrophus collected worldwide revealed polymorphisms in restriction enzyme sites. One such polymorphism, coupled with data on a polymorphism in nuclear rDNA, suggests that there are two genetically distinct but geographically overlapping mating populations of C. heterostrophus in the world.     

A circular 73 kb DNA from the colourless flagellate Astasia longa that resembles the chloroplast DNA of Euglena: restriction and gene map

Summary A 73 kbp circular DNA was isolated from the colourless euglenoid flagellate Astasia longa. Restriction sites of 12 restriction endonucleases were mapped on this DNA. Southern hybridization using plastid gene probes from Euglena, spinach and tobacco revealed sequence homologies to the genes coding for 16S and 23S ribosomal RNAs, elongation factor Tu (tufA) and the large subunit of ribulose-1,5-bisphosphate carboxylase (rbcL). The locations of these sequences on the restriction map were determined. Sequences homologous to chloroplast genes psaA, psbA, psbD, psbE and atpA are not present. The ribosomal RNA genes are organized in three tandem repeats, each containing one 23S and one 16S rRNA gene. In addition, there is one extra 16S rRNA gene. These results indicate the presence of a truncated form of a plastid DNA in Astasia.     

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155

 In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155. Received: 19 September 1995/4 January 1996     

Restriction map of the mitochondrial DNA of the true slime mould,Physarum polycephalum: linear form and long tandem duplication

Summary The mitochondrial DNA (mtDNA) of the true slime mould, Physarum polycephalum strain CH934xCH938, was isolated and characterized by restriction mapping. Cloned fragments of the mtDNA were assembled and used to construct the restriction map. This map showed that the mtDNA was a linear molecule of 86.0 kb with a tandem duplication of 19.6 kb. The terminal fragments were identified by sensitivity to Bal31 exonuclease. One of the duplications was located at the right end and the other was located 5 kb from the left end. Each duplicated segment contained 26 restriction sites for ten enzymes and these restriction sites were completely conserved in each duplication. Genes for the large and small rRNAs were mapped to positions about 30 kb from the right end of the mtDNA by hybridization with its own rRNAs. With the exception of a probe for the gene for the large rRNA in Tetrahymena pyriformis mtDNA, various probes from the mtDNAs of Saccharomyces cerevisiae and T. pyriformis showed no significant hybridization to any of the restriction fragments of the mtDNA from P. polycephalum.     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

Telomeric structures in a linear mitochondrial plasmid from Physarum polycephalum

Summary A mitochondrial plasmid was isolated from Physarum polycephalum and characterized by restriction mapping. Cloned fragments of the plasmid were assembled and used to construct a restriction map. This plasmid was a linear molecule with telomeric structures at each end. Southern hybridization with the ends of the plasmid as probes revealed that the plasmid included repeating units at both ends, with each unit being approximately 125 bp in length. The most extensive array of repeats consisted of at least 17 repetitions of the 125-bp unit. The sensitivity of these repeats to Bal31 exonuclease confirmed that they were at, or very near to, the ends of the plasmid. From the extent of the repetitions, the size of the plasmid was estimated to vary from 13.3 kbp to more than 18.3 kbp.     

Mitochondrial DNA of Schizophyllum commune: restriction map,genetic map,and mode of inheritance

Summary Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4–40) is a circular molecule 49.75 kbp in lenght. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis.     

Physical and gene mapping of cauliflower (Brassica oleracea) mitochondrial DNA

Summary A physical map of the mitochondrial DNA isolated from B. oleracea (cauliflower) inflorescences was constructed with the restriction endonucleases Sall, Kpnl and Bgll. Physical mapping was made using the multi enzyme method with either unlabeled or labeled DNA fragments isolated by preparative electrophoresis and a clone bank prepared by inserting incomplete Sall restriction digests of mitochondrial DNA into a cosmid vector.The different mapping studies led to a circular map, about 217 kb in size, containing the entire sequence complexity of the genome. The 26S and 18S – 5S ribosomal RNA genes appeared to be separated by about 75 kb in this map. However, the particular cross-hybridization between several restriction fragments and the sequential diversity of some cosmids indicated that intra molecular recombination may occur naturally in higher plant mitochondria. Namely, one recombinational event resulted in the ribosomal RNA genes mapping closer together.Abbreviations mtDNA mitochondrial DNA - kb kilobasepairs - rRNA ribosomal RNA - LGT agarose low gelling temperature agarose     

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa

Summary Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3-terminal protion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.     

MboI,ThaI and HinfI endonuclease cleavage maps of the yeast mitochondrial DNA

Summary MboI, HinfI and ThaI cleavage maps have been constructed for the region of the mitochondrial DNA from S. cerevisiae where transfer RNA genes are principally located. About 40 cleavage sites have been localized between the C and P genetic markers. The MboI map covers about 50% of the total mitochondrial genome. For constructing maps we have used a series of rho^– deletion mutants whose mitochondrial DNAs have a typical single deletion structure as judged by previous genetic and physical analyses. The mutant DNAs carry known transfer RNA genes and genetic markers and, therefore, the comparison between genetic and restriction maps has allowed us to localize individual transfer RNA genes within defined physical segments.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA - ThaI formerly TacI     

Restriction map and clone bank of tomato plastid DNA

Summary Tomato plastid DNA has been isolated from leaf chloroplasts andPst1 fragments cloned in the plasmid vector pUC8. Recombinant plasmids containing all but the largestPst1 fragment were identified by colony hybridisation. A restriction map of the plastid DNA for four restriction endonucleases was generated by digestion of the clonedPst1 fragments and by hybridisation to Southern blots. The plastid DNA is a circular molecule of 156.6–159.4 kbp with a large inverted repeat, and shows high conservation of restriction sites with plastid DNA from tobacco andPetunia.     

Molecular cloning and physical mapping of the Neurospora crassa 74-OR23-1A mitochondrial genome

Summary To provide for thorough sampling of the Neurospora crassa mitochondrial genome for evolutionary studies, recombinant plasmids containing each of the EcoRI digestion fragments of the genome were assembled and used to map the locations of 89 additional restriction endonuclease cleavage sites, representing 10 newly mapped enzymes and 2 previously unmapped HincII sites. Data used to locate new restriction sites were obtained from digestions of whole mitochondrial DNA, digestions of the cloned EcoRI mitochondrial DNA fragments and hybridizations between new restriction fragments and the cloned fragments. Length measurements of the total genome and of EcoRI fragment 1 are larger than commonly reported.     

Mapping of mitochondrial DNA from Torulopsis glabrata

Summary Large and small rRNAs have been isolated from mitochondria of the yeast Torulopsis glabrata and have been shown to have lengths of 2,700 bases and 1,400 bases respectively. Construction of a restriction endonuclease site map of mitochondria) DNA has enabled us to position these rRNAs by hybridization of labelled RNA to DNA fragments transferred to nitrocellulose. The large and small mt rRNA genes are separated by a minimum of 1,820 by and a maximum of 2,765 by on the 18,870 by mitochondria1 genome. tRNA genes map within this separating sequence but they are also located distal to both rRNA genes. The implication of these results to the structural relationships of mitochondrial DNAs from yeasts is discussed.     

Physical mapping of genes for chloroplast DNA encoded subunit polypeptides of the ATPsynthase complex from Petunia hybrida

Summary Escherichia coli minicells harbouring the cloned restriction fragment Sall S9 from P. hybrida chloroplast DNA synthesize the beta and epsilon polypeptide subunits of the CF₁ component of the chloroplast ATPsynthase complex. The polypeptides were identified by molecular weight determination and immunoprecipitation. The position of the atpB and the atpE gene, encoding respectively the beta and epsilon subunit, on the Sall S9 fragment was determined in more detail by studying polypeptide synthesis directed by subclones of the S9 fragment in E. coli minicells. The atpB and atpE genes are located close to the rbcL gene, the distance between the rbcL gene and atpB gene being approximately 770 bp. Analysis of the expression of subclones of the S9 fragment in E. coli minicells also revealed that the atpE gene can be transcribed and translated independently of the expression of the atpB gene.The location of the genes coding for the alpha subunit (atpA gene) and the proteolipid subunit III of CF₀ (atpH) of the ATPsynthase complex on the physical map of P. hybrida cpDNA was determined by hybridization of restriction enzyme digests of petunia cpDNA with cloned cpDNA fragments from Spirodela and wheat, containing internal parts of respectively the atpA and the atpH gene. The two genes map close together within a region of 5.2 kbp on the physical map of P. hybrida cpDNA. The distance between the atpA gene and the atpB and atpE genes is approximately 42 kbp.Abbreviations cpDNA chloroplast DNA - rbcL gene coding for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - CF₁ coupling factor, extrinsic part of the chloroplast ATPsynthase complex - kbp kilo base pairs     

Molecular characterization and cloning of sheep mitochondrial DNA

Summary Mitochondrial DNA from the liver of a single Rasa Aragonesa sheep has been isolated and characterized. The size of the genome, determined by restriction enzyme analysis, was found to be 16.58 kbp. The cleavage sites for the restriction endonucleases BamHI, HindIII, EcoRI, BglII, PvuII, BstEII and PstI were mapped, and the gene organization deduced through heterologous hybridization using different cloned fragments of the rat mitochondrial genome. Fragments representative of the entire sheep genome were cloned in plasmid vectors pGEM3Z and pUN121.