The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

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     

The mitochondrial genome of the aquatic phycomycete Blastocladiella emersonii

Summary Mitochondrial DNA from the aquatic fungus Blastocladiella emersonii Cantino and Hyatt has been isolated and characterized. By restriction enzyme analysis the size of the mitochondrial genome was found to be 35.5 kb pairs. A restriction site map was constructed using the cleavage data for 6 endonucleases which showed the mitochondrial genome to be circular. The genes for the small and large ribosomal RNA, the ATPase subunits 6 and 9, the cytochrome c oxidase subunits 1, 2, and 3, and the apocytochrome b were located in the mitochonridal genome of B. emersonii by hybridizations with mitochondrial DNA probes from Saccharomyces cerevisiae and Neurospora crassa     

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.     

Mitochondrial genome of the dimorphic zygomycete Mucor racemosus

Mitochondria were isolated from the dimorphic zygomycete Mucor racemosus by differential centrifugation. DNA from the organelles was purified by cesium chloride-ethidium bromide isopycnic centrifugation. Examination of the mitochondrial DNA by electron microscopy revealed a circular chromosome approximately 63.8 kbp in circumference. The chromosome was digested with restriction endonucleases and the resulting DNA fragments were separated by agarose-gel electrophoresis. Electrophoretic mobilities and stoichiometry of the fragments indicated a mixed population of mtDNA molecules each with a size of about 63.4 kbp. Physical maps were constructed from analyses of fragments generated in single and double restriction digests and from the hybridization of fragments to probes for the large and small mitochondrial rRNA genes from Saccharomyces cerevisiae. The Mucor mitochondrial chromosome was found to exist in the form of two flip-flop isomers with inverted repeat sequences encoding both rRNA genes.     

Organisation of the chloroplast genome of kiwifruit (Actinidia deliciosa)

Summary A restriction map of the chloroplast genome has been determined for kiwifruit, Actinidia deliciosa. Single and multiple enzyme digests of kiwifruit chloroplast DNA were hybridised to a set of Brassica chloroplast probes, and the kiwifruit bands aligned with the known Brassica map. The chloroplast DNA of kiwifruit is typical of the majority of angiosperm chloroplast genomes; it is 160 kb in size, contains a 15–34 kb inverted repeat, and its gene content and gene order are similar to those of the Brassica chloroplast genome.     

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     

Extensive mitochondrial DNA variation in somatic tissue cultures initiated from wheat immature embryos

Summary Wheat mitochondria) DNA has been isolated from callus cultures initiated from both immature embryos and the corresponding parental cultivar. A Sall restriction pattern study has shown that the organization of callus culture mitochondria) DNA underwent extensive change, characterized by either the disappearance or the decrease in the relative stoichiometry of several restriction bands. Hybridization of labelled mitochondrial fragments obtained from a recombinant cosmid library to Southern blots of callus and parental line restricted mitochondria) DNAs has shown that a fraction of the mitochondria) genome was lost in callus cultures. Data from a Sall + HindIII restriction map of a defined part of the wheat mitochondria) genome concerned with some of these variations strongly suggest that the observed variations correspond to the disappearance of at least one mitochondria) DNA subgenomic molecule in callus cultures.Abbreviations mtDNA mitochondrial DNA - cpDNA chloroplast DNA - rRNA ribosomal RNA - mRNA messenger RNA - kb kilobase - cv cultivar     

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.     

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     

Physical mapping of genetic determinants on yeast mitochondrial DNA affecting the apparent size of the Var 1 polypeptide

Summary The var1 locus on yeast mitochondria) DNA is defined by a set of alleles that affect the apparent size (M_r 40,000–44,000) of a mitochondria) translation product (var1), a protein of the mitochondria) 38S ribosomal subunit. Using petite deletion and restriction endonuclease site mapping, we have defined the physical location of all var1 alleles to a specific restriction fragment of roughly 2.1 kbp located between the antibiotic resistance loci ery1 and olil. Surprisingly, from base sequence studies of this region by Tzagoloff et al. (1980), it appears that the DNA fragment we have mapped contains little or none of the structural gene for the var1 protein since the fragment is composed primarily of long stretches of dA + dT interspersed with short clusters high in dG + dC. Nevertheless, by a type of complementation test termed zygotic gene rescue (Strausberg and Butow, 1977), we show that with petites retaining that restriction fragment and short flanking sequences, var1 polypeptide characteristic of the strain from which the petite was derived, is expressed in zygotes formed between the petite and a wild-type tester. Thus the ability of the var1 determinant to act in trans suggests that control of expression of different var1 species involves intermolecular interactions, perhaps at the level of RNA splicing. Our results are discussed in terms of several possible models for the organization and control of the var1 structural gene.     

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.     

The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning,physical mapping and gene location

Summary The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80 500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.     

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.     

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 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     

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.     

Comparison of the genomes of two sympatric iridescent viruses (types 9 and 16)

Summary A map of the sites in the genome ofCostelytra zealandica iridescent virus (CzIV), using the restriction enzymesBamHI,KpnI, andPstI, showed the genome size to be 170.2 kbp in length. It was found that the genome was cyclically permuted and that 39% of the genome of CzIV contained repetitive sequence elements. The genome was found to hybridize with the genome of another iridescent virus, type 9 (WIV), in DNA-DNA hybridization experiments. A region of the WIV DNA genome (23.4 kbp) did not hybridize with CzIV DNA and this region is similar in size to the total genomic size difference between CzIV and WIV (22.4 kbp). A unique repeat sequence from iridescent virus type 6 (CIV) was shown to be present in the genome of WIV but not that of CzIV. Finally, the positions of the major capsid protein genes, VP53 and VP52, in the restriction enzyme maps for type 16 and type 9 respectively were determined.     

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.     

An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeniella genera

Summary Sequences hybridzing to mitochondrial DNA probes from Saccharomyces cerevisiae have been mapped in six mitochondrial genomes from the Dekkera/Brettanomyces yeasts and in mtDNA from the closely related Eeniella nana. Sequence order for the 34.5 kbp mtDNA of E. nana is identical to that for mtDNAs from B. custersianus (28.5 kbp) and B. naardenensis (41.7 kbp) thereby suggesting that the former yeast is affiliated with the latter two species. A closer relationship is suggested for D. intermedia and D. bruxellensis as mtDNAs from these yeasts, 73.2 and 85.0 kbp respectively, have the same sequence order and mostly common restriction endonuclease sites. Differences between the two molecules are reminiscent of those found in mtDNA polymorphisms of S. cerevisiae strains thereby suggesting that the two Dekkera yeasts are variants of a single species. An unusual feature of the Dekkera species mtDNA is an inversion of the cytochrome b hybridizable region relative to the LrRNA sequence. Likewise mtDNA from B. anomalus (57.7 kbp) has an inversion of the cytochrome oxidase subunit 1 sequence with respect to the LrRNA sequence. By contrast the largest mtDNA (101.1 kbp) from B. custersd has the cytochrome b and LrRNA sequences in the same orientation. In addition hybridizable regions in this mtDNA are found in three clusters that are separated by several thousand base pairs of sequence deficient in restriction endonuclease sites. This observation together with the low guanine and cytosine content of the mtDNA suggests that the regions separating the sequence clusters are mostly adenine and thymine residues.