Gene organization of the mitochondrial DNA of yeasts: Kluyveromyces lactis and Saccharomycopsis lipolytica

Summary Restriction fragment maps have been constructed for the mitochondrial DNA from two petitenegative yeasts, Kluyveromyces lactis and Saccharomycopsis lipolytica (Candida lipolytica). On these circular genomes, we localized the sequences homologous to the S. cerevisiae mtDNA fragments carrying known genes. The arrangement of genes for ATPase subunit proteins, ribosomal RNA and 4S RNA shows a common feature with respect to S. cerevisiae mitochondrial genome.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA     

Cytoplasmic male sterility is associated with large deletions in the mitochondrial DNA of two Nicotiana sylvestris protoclones

Summary Two cytoplasmic male-sterile plants (CMSI and CMSII) were obtained by protoplast culture in Nicotiana sylvestris. Both plants showed large deletions (up to 50 kb) in their mitochondrial DNA. Restriction maps of the reorganized regions suggested that the deletions occurred via two homologous recombination events (rec. 1 and rec. 2) in the parental mitochondrial genome. With the exception of nad5, no mitochondrial DNA polymorphism could be detected between parental and CMS lines using different heterologous genes probes. A sequence homologous to the Oenothera nad5 mitochondrial gene was located close to the CMSI-specific rec. 2 region. Moreover, a cDNA probe corresponding to total mitochondrial RNA from the parent line was found to hybridize to mitochondrial DNA fragments involved in the rec. 1 event common to both CMS lines, suggesting that rec. 1 lies in a transcribed region. Cytoplasmic male sterility in the Nicotiana sylvestris CMS mutants could be due either to gene deletion or to a regulatory effect of such a deletion on mitochondrial gene expression, rather than to the presence of specific polypeptides as has been shown in the T cytoplasm of maize, or in CMS Petunia.     

Mitochondrial DNAs of Suillus: three fold size change in molecules that share a common gene order

Summary We constructed restriction-site and gene maps for mitochondrial DNAs from seven isolates of five species of Suillus (Boletaceae, Basidiomycotina). Each mitochondrial genome exists as a single circular chromosome, ranging in size from 36 to 121 kb. Comparisons within species and between two closely related species revealed that insertions and deletions are the major form of genome change, whereas most restriction sites are conserved. Among more distantly related species, size and restriction-site differences were too great to allow precise alignments of maps, but small clusters of putatively homologous restriction sites were found. Two mitochondrial gene orders exist in the five species. These orders differ only by the relative positions of the genes for ATPase subunit 9 and the small ribosomal RNA and are interconvertible by a single transposition. One of the two gene arrangements is shared by four species whose mitochondrial DNAs span the entire size range of 36 to 121 kb. The conservation of gene order in molecules that vary over three-fold in size and share few restriction sites demonstrates a low frequency of rearrangements relative to insertions, deletions, and base substitutions.Abbreviations COI, COII, COIII Cytochrome oxidase subunits I, II, and III - APT6, ATP9; large and small ribosomal RNA gene apocytochrome b, COB; ATP synthase subunits 6, 9 - mtDNA LrRNA, SrRNA; mitochondrial DNA     

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     

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     

Phylogenetic implication of heterogeneity of the nontranscribed spacer of rDNA repeating unit in various Neurospora and related fungal species

Summary Nontranscribed spacer (NTS) regions of ribosomal (r)RNA genes are non-conserved and are shown to be useful for phylogenetic studies. ³²P-labelled N. crassa NTS pCC3400 DNA, was used as a molecular probe to hybridize Southern blots of genomic DNAs obtained from Neurospora, Gelacinospora, Sordaria, bacteria, plants, and animals. Our studies conclude that: (a) the homotahllic species of Neurospora should not belong to genus Gelacinospora (a historical question) and that Neurospora homothallic species are closer to Gelacinospora than to Sordaria; and that (b) all of the filamentous fungal species tested are indeed closer to the higher plant genome than to higher primate animal genome based on shared restriction sites of 12 enzymes. Our studies also demonstrate the usefulness of nontranscribed rRNA gene probes in resolving questions regarding phylogenetic relatedness between widely separated organisms using the parsimony principle based on mutation sites from DNA restriction maps; it has not been possible to do this using DNA: DNA hybridization procedures that involved the total genome.     

Physical organization of the 18S and 5S ribosomal RNA genes in the mitochondrial genome of rye (Secale cereale L.)

Summary The mitochondrial 18S and 5S ribosomal RNA (rRNA) genes of rye, plus a total of about 90 kilobase pairs of flanking DNA, have been cloned and maps of restriction enzyme cleavage sites have been constructed. Like their homologs from hexaploid wheat, the rye genes are closely linked and are part of a three-copy family of recombining repeats (the 18S/5S repeat). The rye repeat probably also contains a mitochondrial tRNA^fMet gene, which the wheat repeat is known to carry. However, despite the overall organizational similarity between the wheat and rye 18S/5S repeats in the immediate vicinity of their coding regions, extensive rearrangement of flanking sequences has taken place during evolutionary divergence of the two species. Our data provide additional support for an emerging picture of plant mitochondrial genomes as evolving much more rapidly in structure than in sequence.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe

Summary The three mutator strains ana ^r-8, ana ^r-14, and diu ^r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe. Abbreviations. MtDNA = mitochondrial DNA; S. pombe = Schizosaccharomyces pombe; cox1, cox2, and cox3 refer to the mt genes coding for the three subunits of cytochrome oxidase; ATPase 6 (oli2), ATPase 8 (aapl in Saccharomyces cerevisiae, urf a61 in HeLa) and ATPase 9 (olil) refer to the three respective subunits of ATP synthase; cob is thegene for apocytochrome b; urf a is the single intergenic unassigned reading frame in S. pombe; 1 rRNA and s rRNA refer to the large and small ribosomal RNA, respectively. Mut^– is a cytoplasmic mutator (the corresponding wild type allele is mut⁺). Mit^– are mitochondrially inherited respiratory deficient mutants with mitochondrial protein synthesis; RC = respiratory competent, RD = respiratory deficient.     

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.     

Yme2p is a mediator of nucleoid structure and number in mitochondria of the yeast Saccharomyces cerevisiae

A large number of gene products have been identified that either directly or indirectly alter the inheritance of mitochondrial DNA. In yeast, we have used a unique genetic screen based on the transfer of DNA from mitochondria to nucleus to identify nuclear-encoded gene products that are targeted to mitochondria and impact the stable inheritance of mitochondrial DNA. A specific allele of one of these genes, yme2-4, prevents even the low wild-type rate of mitochondrial DNA transfer to the nucleus and imparts significant temperature-sensitive and respiratory-growth defects. Intra- and extragenic suppressors of the yme2-4 growth phenotypes were isolated and analysis of these interacting genes reveals that both YME2 and its suppressors influence the structure and number of mitochondrial nucleoids. The yme2-4 allele decreases the average number of mtDNA nucleoids found in cells and the sensitivity of DNA in toluene-treated mitochondria to digestion by DNA exonuclease, effects reversed by intra- and extragenic suppressors. The extragenic suppressor, a missense allele of ILV5, encodes an enzyme of the branched-chain amino acid biosynthetic pathway that is also a component of mitochondrial nucleoids. A null allele of ILV5 suppresses transfer of mitochondrial DNA to the nucleus and displays synthetic interactions with yme2-4.     

Mitochondrial DNA polymorphism in the Vietnamese population

The mitochondrial DNA variation was screened in a sample of 50 unrelated individuals of the Vietnamese population originating from Hanoi. A combination of long and standard PCR and restriction endonuclease digests with the enzymes HpaI, BamHI, HaeII, MspI, AvaII and HincII were used to reveal mtDNA variation. Twenty enzyme morphs were detected, three of which (HaeII-13^Viet, MspI-19^Viet and MspI-20^Viet) are new and are produced by a single mutational event in already known enzyme morphs. Ten already known and four new mitotypes [93^Viet (1-1-2-4-1), 94^Viet (2-1-13^Viet-1-1), 95^Viet (2-1-13^Viet-19^Viet-1) and 96^Viet (1-1-2–20^Viet-12)] were found in the Vietnamese population. The 9-bp deletion occurring in the COII/tRNA^Lys region of the mitochondrial genome was also analysed and 10 samples were found to have this deletion. The comparison of the Vietnamese with other East Asian populations showed a close genetic relationship of the population under investigation with other Orientals. However, the Vietnamese population can be differentiated by the significantly higher frequency of the enzyme morph HincII-5 and by seven new markers. These results strongly support the hypothesis of a dual ethnic origin of the Vietnamese population from the Chinese and Thai–Indonesian populations based on HLA markers and linguistic evidence.     

Fragmentation of bacteriophage S13 replicative form DNA by restriction endonucleases from Hemophilus influenzae and Hemophilus aegyptius.

The restriction enzymes Hind from Hemophilus influenzae and Hae_III from Hemophilus aegyptius cleave bacteriophage S13 replicative form (RF) DNA into 13 and 10 specific fragments, respectively. The sizes of these fragments were estimated by gel electrophoresis, electron microscopy, and pyrimidine isostich analysis. The Hind and Hae_III fragments were ordered relative to each other by cross digestion and a physical map of the S13 genome constructed. Comparison of the Hind cleavage patterns of S13 RF DNA and ?X174 RF DNA showed the majority of the fragments from the two DNAs coincided with each other except for three of the thirteen S13 fragments and three of the thirteen ?X174 fragments. Comparison of the Hae_III patterns of the two DNAs revealed a lack of coincidence of one S13 fragment only and two ?X174 fragments. From the data obtained by the cleavage of the two DNAs by Hind and Hae_III, a correlation between the physical restriction enzyme cleavage maps and the genetic map of the two phages was made. The differences in cleavage of the two DNAs by the restriction enzymes have been explained by changes in two restriction enzyme sites in the AB region and one change of site in the F region of the genetic map of the two bacteriophages.     

Neurospora crassa nuclear genome contains analogy of Saccharomyces cerevisiae genes for ribosomal RNA processing

Summary Neurospora crassa wild type genome shows DNA sequences which are homologous to the sequences present in the rRNA processing genes of the yeast Saccharomyces cerevisiae. Five such processing genes from yeast, viz., RNA1 through RNA5, cloned in plasmid pBR322 were transformed in Escherichia coli strain LE392. Southern blots containing DNAs from these clones were restricted with several restriction endonucleases along with DNAs from lambda phage, rice (plant) and neuroblastoma (animal), were hybridized with ³²P-labelled nick-translated N. crassa nuclear DNA under very stringent conditions. Autoradiograms of these blots revealed that four yeast rRNA processing genes (RNA1, RNA2, RNA3, and RNA4) showed homology with N. crassa nuclear DNA but such analogs were not found in DNAs representing prokaryotes, phages, higher plants and animals.     

Orthopoxvirus DNA: A comparison of restriction profiles and maps

Characteristic DNA endonuclease digest fragment electropherograms and restriction site maps permitted differentiation and genome structure analysis of 38 orthopoxviruses that included isolates of monkeypox virus from humans and animals, monkeypox white variants, variola, vaccinia, ectromelia, Tatera (gerbil) and raccoon poxviruses, and cowpox and camelpox viruses. HindIII cleavage sites mapped on the 38 virus genome DNAs plus SmaI, BglI, SacI, KpnI, XhoI, and SalI maps for variola (Harvey) and monkeypox (Copenhagen) virus DNAs were derived essentially by cross-hybridizations with monkeypox, vaccinia, and variola virus-cloned DNA restriction fragments, thus digest fragments could be assigned homologous regions on previously established genome maps. Salient of our observations, the DNA HindIII maps correlated to a high degree, but variations in middle and especially terminal DNA region cleavage sites provided a basis for discerning species, strains and variants. The extent of the inverted terminal repetitions (ITRs) for 37 DNAs were determined with HindIII, PvuI, SalI, and ClaI, plus nine more restriction enzymes for Bangladesh variola virus DNA by hybridizations with either the terminal tandemly repeated 70-bp segment or an EcoRI-PvuI near hairpin-end 75-bp segment from WR vaccinia virus. The opposite terminal regions of variola DNA were considerably asymmetrical compared to the large symmetrical ITRs of the other species examined. An apparent DNA inversion and concurrent deletion (1 kbp) with subsequent repair of DNA to original structure was suggested from right terminal region maps of four viruses chosen from a variola virus passage series in monkeys. Correlative with virus geographic distribution, two strains of monkeypox virus, each containing two variants, were differentiated by DNA profiles of isolates from smallpox-like disease (SLD) patients of the African rainforest region. The DNAS of five monkeypox viruses isolated from laboratory and zoo animals resembled most DNAs from SLD monkeypox viruses from Sierra Leone. A poxvirus from an American raccoon contained 40% DNA that did not cross-hybridize with orthopoxvirus DNA probes. The DNAs of recent isolates from a gerbil and from a camel each mapped as unique African orthopoxvirus species and differed from variola virus.     

A possible effect of viral proteins on the specificity of interference by defective vesicular stomatitis virus particles

By hybridizing individual early RNA species from T7-infected cells to HpaI cleavage products of T7 DNA, we have identified the fragments which comprise the early region of the T7 genome and have deduced their order with respect to the genetic and physical maps. From the left end of the DNA molecule the arrangement of HpaI DNA fragments is: F·H·G·Q. Our results indicate that the termination signal for the host RNA polymerase is situated in, or near the right end of, HpaI fragment Q (at about 19.3% of the genome). In vivo, recognition of the termination signal at the end of the early region by the host RNA polymerase occurs with about 70–75% efficiency. Late mRNA from T7-infected cells was also analyzed by this technique. Our results demonstrate that both class II and class III genes are efficiently transcribed during the interval from 6 to 12 min after infection. These results are contrasted with those observed for the purified phage RNA polymerase in vitro.     

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.     

Polymorphism of the mitochondrial L-RNA gene of the basidiomycete Coprinus cinereus

Summary Two unexpectedly small mitochondrial (mt) genomes of Coprinus cinereus, P and S, were compared with the H and J genomes we have described previously. H and J are 42 kb in size and differ in having alternative 1.23 kb insertions in or adjacent to the co-1 gene. P and S DNAs lacked both insertions and had an identical 4.4 kb deletion between the co-1 and L-RNA gene. P DNA contained a 700 by insertion and S DNA a 300 by deletion within a sequence coding the L-RNA gene. This was shown by Southern blot analysis using probes containing the 5 or the 3 exon sequences of the L-RNA gene of Neurospora crassa. These hybridisations showed also that the L-RNA gene and co-1 gene in the C. cinereus mt genome are oppositely orientated and must be transcribed from different DNA strands. No DNA homology was detected using probes containing intron sequences from the L-RNA genes of Saccharomyces cerevisiae or N. crassa. There was no evidence of respiratory deficiency in P and S strains and transfer of nuclei by dikaryon formation made it possible to recombine H nuclei with P and S mitochondria, S nuclei with H and P mitochondria and P nuclei with H mitochondria with no apparent detrimental effect on growth. We conclude that P and S mtDNAs represent naturally occurring variants of the C. cinereus mt genome.     

Transformation ofNeurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA

Summary We have conducted a detailed study of 108qa-2 ⁺ Neurospora transformants which were obtained by use of circular plasmid DNAs and various linear DNAs. Parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which theqa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass theqa-2 gene through a cross. The non-transmissible class comprises the majority of transformants and may identify those which harbor autonomously replicating plasmids. Evidence is presented which suggests that a 1.2 kB BamHI-Bg1IIqa-2 ⁺ DNA fragment might possess anars sequence. Transformation with linear plasmid DNAs and DNA fragments carrying theqa-2 gene resulted in a demonstrable increase in transformation frequency beyond that achieved with circular plasmid DNAs, but did not permit precise targeting to the resident locus. Southern analysis showed that linked transformants have only the normal residentqa-2 band whereas the unlinked transformants always possess the resident band plus at least one additional band. Multiple integration events appear to be common and include cases where only a portion of the transforming DNA has been integrated.     

Genetic maps of eight linkage groups of Aspergillus niger based on mitotic mapping

Summary This paper provides a genetic map of Aspergillus niger. At present 84 markers have been assigned to eight linkage groups. The chromosomal location of 60 markers is presented in this paper. The allocation of markers is based on recombination due to mitotic crossing over. Various methods for selection and analysis of homozygous recombinants were applied, using colour, auxotrophic and resistance markers. In addition, transformants carrying the heterologous Aspergillus nidulans gene coding for acetamidase (amdS) were used for mitotic mapping of markers in several linkage groups. In most of the transformants the amdS insert appeared to be centromere-distal to all known genetic markers, thus extending the eight linkage groups has been determined. On the basis of these and earlier experiments tentative genetic markers were found on both arms of the chromosomes, except for chromosomes II and IV. The genetic distance between markers and the centromere varies from about 10^-4 (LG I, II, V) up to more than 10^-2 (LGIII, VI, VIII). The total frequency of mitotic recombination per genome in this fungus has been estimated to be at least 1.2×10^-1.     

Inheritance of mitochondrial and chloroplast genome markers in backcrosses of Chlamydomonas eugametos x Chlamydomonas moewusii hybrids

Summary Southern blot analysis of AvaI-digested total cellular DNA from the interfertile species Chlamydomonas eugametos and Chlamydomonas moewusii with a coxI mitochondrial gene probe from Chlamydomonas reinhardtii revealed single hybridizing fragments of 5.0 and 3.5 kb, respectively. The transmission of these mitochondrial DNA physical markers along with that of chloroplast genetic markers for resistance to streptomycin and resistance to erythromycin was studied in the fourth backcrosses of F₁ hybrids to one or the other parent. Viability in these backcrosses is high in contrast to the cross C. eugametos x C. moewusii and its reciprocal which are associated with considerable meiotic product lethality. The resulting zygospores were found to transmit the mitochondrial and chloroplast genome markers uniparentally or preferentially from the mating-type-plus parent. Thus the species pair C. eugametos and C. moewusii differs from the pair Chlamydomonas reinhardtii and Chlamydomonas smithii in which mitochondrial genome markers are transmitted uniparentally by the mating-type minus parent, while the chloroplast genome markers are transmitted uniparentally by the opposite parental mating-type (Boynton et al. 1987).