Intron mobility results in rearrangement in mitochondrial DNAs of heterokaryon incompatible Aspergillus japonicus strains after protoplast fusion.

Mitochondrial transmission was carried out under selective conditions between incompatible Aspergillus japonicus strains always using an oligomycin-resistant mitochondrial donor and selecting for recipient nuclei and oligomycin-resistant mitochondria. All attempted intraspecific mitochondrial transmissions were successful, but the transmission between closely related A. japonicus and A. aculeatus failed. Under selection pressure, resistant progeny harbor the mitochondrial DNA (mtDNA) of the donor strain, which may remain unchanged or may be modified by the introns of the recipient mitochondrial genome. Detailed analysis of a certain strain harboring rearranged mtDNA suggests that the mtDNA profiles of recombinant-like progeny are strongly influenced by the characteristics and mobility of introns of both parental mtDNAs. Both intron loss and intron acquisition play a role in the rearrangement of mtDNA. In certain parental combinations, a particular intron was lost very frequently.     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f ). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data. Received: 16 July 1996 / Accepted: 2 December 1996     

Recombination of mitochondrial DNAs following transmission of mitochondria among incompatible strains of black Aspergilli

Successful intra- and interspecific mitochondrial transfers were performed by polyethylene glycol (PEG)-induced protoplast fusion among incompatible strains belonging to the Aspergillus niger species aggregate. The mitochondrial DNAs (mtDNAs) of the strains examined were of three main types based on their restriction fragment length polymorphism (RFLP) profiles. mtDNA types 1 and 2 correspond to A. niger and A. tubingensis species, respectively, while type 3 is represented by some Brazilian wild-type isolates (possibly a distinct species or subspecies). mtDNA types 1 and 2 could be further divided into several subgroups (1a–1e and 2a–2f?). All these strains, representing different RFLP groups or subgroups, were fully incompatible with respect to nuclear complementation. The transfer experiments were carried out under selection pressure, using a mitochondrial oligomycin-resistant mutant of mtDNA type 1a as donor. Following fusion mitochondrial oligomycin-resistant progenies were recovered in the presence of oligomycin by selecting for the nuclear phenotypes of the oligomycin-sensitive recipient strains. All attempted transfers were successful, and resulted in different varieties of resistant recombinant mitochondrial progenies at various frequencies. Within the group of strains of mtDNA type 1, the transfer of oligomycin-resistant mitochondria resulted in the appearance of a single recombinant type of RFLP profile in each case. The recombination events were more complex when the transfer of oligomycin resistance occurred between strains representing different species (mtDNA groups 1a→2 and 1a→3). A great variety of recombinant mtDNA RFLP profiles appeared. Explanation for this phenomenon are discussed on the basis of preliminary physical mapping data.     

The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome

Summary In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain ana ^r -14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome - c oxidase - cob gene encoding apocytochrome b - cox1I1, cox1I2a, cox1I2b, cox1I3 introns in cox1 - cox1Ix ^+/– indicates the presence or absence of the intron either in the native gene or after intron DNA excision - cox1Ix is a deletion in the intron leading to respiratory deficiency     

Maternal inheritance of mitochondria in Eucalyptus globulus

It is important to verify mitochondrial inheritance in plant species in which mitochondrial DNA (mtDNA) will be used as a source of molecular markers. We used a polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) approach to amplify mitochondrial introns from subunits 1, 4, 5, and 7 of NADH dehydrogenase (nad) and cytochrome oxidase subunit II (cox2) in Eucalyptus globulus. PCR fragments were then either sequenced or cut with restriction enzymes to reveal polymorphism. Sequencing cox2 showed that eucalypts lack the intron between exons 1 and 2. One polymorphism was found in intron 2-3 of nad7 following restriction digests with HphI. Fifty-four F1 progeny from seven families with parents distinguishable in their mitochondrial nad7 were screened to show that mitochondria were maternally inherited in E. globulus. These results constitute the first report of mitochondrial inheritance in the family Myrtaceae.     

Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori sequences are deleted

Summary Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.This paper is dedicated to colleagues J. Jana, D. Tasi, I. Bortner, and F. Zavrl     

Two homologous mitochondrial introns from closely related Saccharomyces species differ by only a few amino acid replacements in their Open Reading Frames: one is mobile, the other is not.

We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis. The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns). We have shown that the S. capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S. cerevisiae intron is not. Determination of sequence of the S. capensis intron demonstrates that it differs from that of the homologous S. cerevisiae intron (bi2) by a very small number of nucleotide substitutions.     

Construction of novel cytochrome b genes in yeast mitochondria by subtraction or addition of introns

The mitochondrial cob-box gene coding for apocytochrome b in yeast has five introns and six exons or two introns and three exons depending on the wild-type strain considered. Some intron mutations in this gene affect not only its expression but also that of another mitochondrial gene: oxi3. To understand better the function of introns in gene expression, we have constructed a series of new strains that differ only by the presence or absence of one of the five wild-type introns in the cytochrome b gene, the rest of the mitochondrial and nuclear genome remaining unchanged. All constructions result from in vivo recombination events between rho- donor and rho+ recipient mtDNA. The following genes have been constructed: [see text]. Interestingly, all the genes lead to the synthesis of cytochrome b, while only the genes having the intron bI4 allow the expression of oxi3. A nuclear gene, when mutated, can compensate for the absence of the intron bI4.     

Genetic variation in cultivated strains of <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Genetic differences among Agaricus blazei strains were investigated using somatic incompatibility testing, isozyme analysis, restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA), and random amplified polymorphic DNA (RAPD) analysis. Eight strains, one cultivated strain from Brazil and seven from Japan, were used in this study. Somatic incompatibility interactions were observed between the Brazilian cultivated strain and the Japanese strains. The Brazilian cultivated strain had its own distinct patterns of esterase isozyme and mtDNA RFLP, but all seven Japanese cultivated strains showed identical patterns. When the RAPD patterns, obtained using eight primers, were compared the eight strains had their own distinct RAPD profiles. Distance values were calculated between all pairs of the strains based on presence or absence of individual RAPD bands, and a dendrogram was constructed by unweighted pair-group method with arithmetic clustering (UPGMA) analysis. Seven Japanese cultivated strains were grouped to each other, and this group was finally linked to the Brazilian cultivated strain. Based on these results, the degree of genetic variation among the A. blazei strains used is discussed.     

Analysis of chloroplast and mitochondrial DNAs in asymmetric somatic hybrids between tobacco and carrot

Summary Chloroplast and mitochondrial DNAs have been examined by comparison of restriction enzyme patterns in asymmetric hybrid plants, resulting from the fusion between leaf mesophyll protoplasts of Nicotiana tabacum (Solanaceae), and irradiated cell culture protoplasts of Daucus carota (Umbellifereae). These somatic hybrids with normal tobacco morphology were selected as a consequence of the transfer of methotrexate and 5-methyltryptophan resistance from carrot to tobacco. The restriction patterns of chloroplast DNAs in somatic hybrids were indistinguishable from the tobacco parent. However, we found somatic hybrids with mitochondrial DNA significantly different from either parent, as judged by analysis of fragment distribution after restriction enzyme digestion. The possible formation of altered mitochondrial DNA molecules as the result of parasexual hybrid production between two phylogenetically highly divergent plant species will be discussed.     

Interpretation of variability of mitochondrial genomes in the species Aspergillus carbonarius

For interpretation of intraspecific polymorphism and the considerable differences in the size of mtDNAs among three groups of A. carbonarius, restriction maps were constructed from several enzymes. Functional maps were also developed to compare genome organisations and gene content. The appearance of various mtDNAs of A. carbonarius strains are different in size, but their gene content is almost identical. The 1.1 kb size difference between two closely related subgroups (1a, 1b) can be attributed to the presence or absence of an intron in cox2 gene. This phenomenon demonstrates that the migration of introns is possibly responsible for the development of variable mitochondrial genomes in nature. The striking differences in size and restriction patterns between two main mtDNA groups might derive from both the intronal variations and the altered intergenic organisation.     

Presence of interaction but not complementation between human mtDNAs carrying different mutations within a tRNA(Leu(UUR)) gene

This study provided answers to fundamental questions on mammalian mitochondrial genetics: Could respiratory function in mitochondria be maintained by their exchange of genetic contents even when mutations were created within the same genes in different mitochondrial DNA (mtDNA) molecules? Using cell fusion techniques, we created a chance to coexist two types of respiration-deficient syn(-) mitochondria carrying different mtDNA mutations within the same tRNA(Leu(UUR)) gene obtained from patients with mitochondrial diseases. The results showed that two syn(-) mitochondria exchanged their genetic contents, but did not restore respiration defects, suggesting that mitochondrial interaction could not complement the mutations created within the same gene in different mtDNA molecules.     

Restriction Enzyme Analysis of Mitochondrial DNA of Acanthamoeba Strains in Japan

ABSTRACT. Eight isolates, identified as either Acanthamoeba castellanii or A. polyphaga from human eye infections, contact lens containers, and soil in Japan, were characterized by restriction fragment length polymorphisms (RFLP) of mitochondrial DNA (mtDNA). Mitochondrial DNA was digested with either Bgl II, Eco R I, Hind III, Hpa I, Sca I or Xba I, electrophoresed in agarose gels, and stained with ethidium bromide. Four distinct RFLP phenotypes that refer to the collection of six fragment size patterns obtained for a single strain with six enzymes, were discovered among the eight strains used in this study. Three strains morphologically classified as A. polyphaga share a single RFLP phenotype with the Ma strain of A. castellanii. The interspecific sequence differences of 7.06–12.74% in DNA nucleotide were estimated from the proportion of DNA fragments shared by each pair of mtDNA.     

Heritable somaclonal variation in wild barley (Hordeum spontaneum)

Summary Progenies of H. spontaneum plants regenerated from immature embryo derived calli were analysed for somaclonal variation on the following traits: (1) organization of the intergenic spacer of the rRNA genes; (2) B and C hordein pattern on SDS-PAGE; (3) genomic organization of the B and C hordein coding sequences; (4) mitochondrial DNA organization assayed by hybridization of Southern blots of total DNA with mitochondrial coding genes; (5) cytology. One out of twelve progeny plants was characterized as variant for two traits: (a) a loss of 1.8 and 2.5 kb Taq I intergenic rDNA spacer fragments and (b) a variant pattern of hordeins on 1-D SDS-PAGE. No numerical or structural chromosome variation was detected among the control plants therefore it is assumed that the variation was caused by the in vitro culture and transmitted, through sexual reproduction, to the analysed progeny.     

Inheritance of mitochondrial DNA in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis>

By crossing Brachionus plicatilis s.s. NH1L strain and German strain, we obtained two types of hybrids, NH1L female × German male designated as NXG and German female × NH1L male designated as GXN. To confirm the crossing of the two hybrid strains at the genetic level, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis using 10 kinds of primers (10 and 12 mers) was carried out. Some amplified DNA fragments from RAPD of the hybrid strain showed mixed patterns of both parental strains, thus confirming that both hybrids were crossbreeds of the NH1L and German strains. Using these hybrids, we investigated the mode of mitochondrial inheritance in B. plicatilis. Full-length mtDNA of the four strains was amplified by PCR, and digested with restriction enzymes to obtain restriction fragment length polymorphism (RFLP) patterns. Both hybrid strains had the same RFLP patterns as their female parents. This result shows that mitochondrial inheritance in rotifers is maternal. Guest editors: S. S. S. Sarma, R. D. Gulati, R. L. Wallace, S. Nandini, H. J. Dumont and R. Rico-Martínez Advances in Rotifer Research     

Further evidence for a high position-specific effect in the action of chemical mutagens on the chromosomes of barley

Summary B1 and B2 are small, circular, mitochondrial plasmid-like DNAs found in male-sterile cytoplasm (cms-Bo) of rice. In this study, nuclear sequences homologous to these DNAs were investigated among a number of rice cultivars. Several copies of nuclear B1-and B2-homologous sequences were detected in all examined cultivars, regardless of the presence or absence of the B1 and B2 DNAs in mitochondria, indicating that the existence of the B1- and B2-homologous sequences in the rice nuclear genome was widespread. A restriction fragment length polymorphism (RFLP) was detected for both sequences, and we propose that these DNAs could be useful RFLP markers for the rice nuclear genome. To analyze these nuclear homologues genetically, segregation analysis of the RFLP was carried out in the F₂ progenies of an Indica-Japonica rice hybrid. Of the B1 homologues, there were two nonallelic fragments, one specific to the Indica parent and the other to the Japonica. These results indicate that the B1 and B2 homologues were dispersed in the nuclear genome. The integration of B1-homologous DNA into the nuclear DNA may have occurred independently after sexual isolation of the Indica and Japonica rice varietal groups, or a intranuclear transposition of these sequences took place during the process of rice differentiation into the varietal groups.     

Simulating the mitochondrial DNA inheritance

Summary Analysing the current mitochondrial DNA patterns biologists have concluded that we all descend from the same mitochondrial Eve, who is postulated to have lived around 200.000 years ago. Such a result is in agreement with the coalescence theory. Here we represent the mitochondrial DNAs as bitstrings that are maternally transmitted with mutations, and that may also participate in the selection process for survival together with the nuclear DNAs. We end up with the same common ancestor, whose mitochondrial DNA can be traced back from the current population, despite the mitochondrial mutations considered. For a given mutation rate, the degree of confidence of this tracing-back process increases even further when the selection mechanism is included.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms