Mitochondrial Genome Size Research Articles

25] Genetics and transformation of mitochondria in the green alga Chlamydomonas

This chapter discusses genetics and transformation of mitochondria in the green alga Chlamydomonas. Molecular evidence strongly supports the endosymbiotic origin of mitochondria from alpha purple bacteria. Photosynthetic eukaryotes show the greatest diversity in mitochondrial genome size and structure, ranging from the 15-kb linear genome of the green alga Chlamydomonas reinhardtii to genomes of certain angiosperms that exceed one Mb. Although the mitochondrial genomes of numerous invertebrates and vertebrates and the liverwort Marchantia is completely sequenced, and the genes they encode are well characterized, the genetic systems in these organelles are poorly developed. Mitochondria of animal cells lacking DNA can be obtained by prolonged culture of cells in medium containing ethidium bromide, and pyruvate and uridine, which are essential for growth of the cell. Genetic dissection of mitochondrial structure and function in baker's yeast is possible because mitochondrial function is dispensable. Mitochondrial transformation in yeast is just beginning to be exploited to study expression of specific organelle genes, and much needs to be done to take advantage of this technique in Chlamydomonas.

Purification of mitochondria and mitochondrial nucleic acids from embryogenic suspension cultures of a gymnosperm, Larix x leptoeuropaea

After a number of attempts to isolate mitochondria from different conifer tissues, embryogenic suspension cultures of hybrid larch (Larix x leptoeuropaea) were developed which enabled the purification of mitochondria using slight modifications to standard techniques. The mitochondrial purity was verified by analysis of the mitochondrial RNA, DNA and proteins. The larch mitochondrial genome size is surprisingly large (> 1000 kbp) and the polypeptide pattern differs greatly from those of wheat or potato mitochondria, suggesting that valuable evolutionary insights will be gained from comparisons between gymnosperm and angiosperm mitochondria. The ease with which embryogenic conifer suspensions can be initiated and used for mitochondrial purification implies that they will be the material of choice for future studies of this type.

Endotherms, ectotherms, and mitochondrial genome-size variation

The patterns of mitochondrial genome-size variation were investigated in endothermic and ectothermic species to examine the role that thermal habit might play in the evolution of animal mitochondrial DNA (mtDNA). Data on mtDNA size (the modal, largest, and smallest mtDNA reported within a species), the percent variation in mtDNA size (the difference in size between the largest and smallest mtDNAs divided by the model genome size for a given species), and the frequency of heteroplasmic individuals (those carrying more than one mtDNA length variant) were tabulated from the literature. Endotherms showed significantly less variation in mtDNA size and tended to have smaller mtDNAs than ectotherms. Further comparisons between endothermic and ectothermic vertebrates revealed that the largest genome and the percent variation in genome size were significantly smaller in the former than the latter. There was no difference between endotherms and ectotherms in the frequency of heteroplasmy. These data are discussed in light of two hypotheses: (1) more intense directional and purifying selection for small genome size in the cytoplasms of species with higher metabolic rates and (2) reduced mutation pressures generating mtDNA size variants in endotherms relative to those in ectotherms. The general trends are consistent with the selection hypothesis but in certain species mtDNA size variation appears to be governed by mutational pressures. To test these competing hypotheses further, comparative studies are proposed where mitochondrial genome size is quantified in sister taxa and tissue types with very different metabolic rates.

DNA ANALYSIS OF EUKARYOTIC ALGAL SPECIES1

ABSTRACTPlastid genomes of algae resemble those of terrestrial plants in form, size, and rate of nucleotide sequence change. They are circular and range in size from 73 kilobases (kb) to over 400 kb. Their many copies per cell can compose >15% of total cell DNA. Mitochondrial genomes, like plastid genomes, are present in high copy number in preparations of total algal cell DNA. Almost all known algal mitochondrial DNA genomes are relatively small, <50 kb; in some species they are linear, whereas in others they are circular.One of the persistent perplexities for phycologists is the question of what relationship two clones or two groups of organisms bear to each other. Several relatively simple techniques can reveal whether or not two organisms belong to the same clone. Total mitochondrial genome size can be compared directly between isolates, although identity in size does not necessarily mean identity in sequence. Restriction endonuclease digestion combined with probing permits comparison of DNA fragment patterns to see if there is identity or near identity between two samples. This methodology can be applied both to organelle genomes and to nuclear genomes.So far, restriction endonucleases cleave plastid and mitochondrial DNA of organisms belonging to the same gene pool into nearly identical fragment patterns, whereas organisms nearly or totally incapable of interbreeding display patterns wherein ≫ 50% of restriction fragments differ in position on an agarose gel after electrophoresis. Thus, organelle genomes may be the first choice for comparing both total size and restriction endonuclease fragment patterns to obtain an indication of whether two organisms are closely related. This methodology can be applied both to organisms in which interbreeding is easy to test and to the many algae in which homothallism or lack of sexual clones has precluded standard breeding analyses. With further data on variability levels within and between fertile populations, it may be possible to state with confidence whether a sample of morphologically similar organisms shares a common gene pool, even if their breeding cannot be manipulated experimentally.

Fluorescence microscopic study of the formation of giant mitochondrial nuclei in the young ovules ofPelargonium zonale

The size of mitochondrial genomes in higher plants are known to range from 200 to 2400 kilobase pairs. However, we failed to identify cytochemically any mitochondria that contain an identifiable master mitochondrial genome. In the present experiments, we have found the giant mitochondrial nuclei which have the capacity for including the master mitochondrial genome in the young ovaries ofPelargonium zonale by use of a 4′-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy, a Technovit embedding, and a video-intensified photon counting system.

Mitochondrial DNA evolution in theobscura species subgroup ofDrosophila

Mitochondrial DNA (mtDNA) restriction site maps for nine species of the Drosophila obscura subgroup and for Drosophila melanogaster were established. Taking into account all restriction enzymes (12) and strains (45) analyzed, a total of 105 different sites were detected, which corresponds to a sample of 3.49% of the mtDNA genome. Based on nucleotide divergences, two phylogenetic trees were constructed assuming either constant or variable rates of evolution. Both methods led to the same relationships. Five differentiated clusters were found for the obscura subgroup species, one Nearctic, represented by Drosophila pseudoobscura, and four Palearctic, two grouping the related triads of species Drosophila subobscura, Drosophila madeirensis, Drosophila obscura, Drosophila subsilvestris, and two more represented by one species each, Drosophila bifasciata, and Drosophila tristis. The different Palearctic clusters are as distant between themselves as with the Nearctic one. For the related species D. subobscura, D. madeirensis, and D. guanche, the pair D. subobscura-D. madeirensis is the closest one. The relationships found by nucleotide divergence were confirmed by differences in mitochondrial genome size, with related species sharing similar genome lengths and differing from the distant ones. The total mtDNA size range for the obscura subgroup species was from 15.5 kb for D. pseudoobscura to 17.1 for D. tristis.

Mitochondrial DNA of Physarum polycephalum: Physical mapping, cloning and transcription mapping

Mitochondrial DNA (mtDNA) has been isolated from four strains of Physarum polycephalum and a restriction site map has been determined using nine restriction enzymes. The restriction site maps of the four strains are similar but each strain is distinguished by insertions, deletions and restriction enzyme site polymorphisms. The sum of the restriction fragments gives mitochondrial genome sizes which vary from about 56 kb to 62 kb. In all four strains the composite map of the restriction enzyme sites for the mtDNA is circular. Knowledge of the restriction enzyme map has enabled cloning of mtDNA fragments representing the entire mtDNA of strain M3. The cloned fragments have been used to create a transcription map of the mtDNA.

Kinetoplast DNA-aggregating proteins from the parasitic protozoan Crithidia fasciculata

Proteins from C. fasciculata, which preferentially aggregate the parasite's AT-rich mitochondrial (kinetoplast) DNA in vitro, are reported. The aggregation is non-topological, non-covalent, independent of Mg 2+ and ATP, but much improved by spennidine. We discuss how these proteins might modulate the shape and size of the parasite's mitochondrial genome.

The linear 20 kb mitochondrial genome of Pandorina morum (Volvocaceae, Chlorophyta)

A physical restriction map of the mitochondrial genome from one clone (TCC 854) of the sexually isolated populations (syngens) of the morphologically uniform species Pandorina morum Bory has been constructed using restriction endonucleases Ava I, Bam HI, Bgl II, Eco RI, Kpn I, and Pst I. The 20 kb linear genome can easily be separated from plastid DNA, nuclear satellite rDNA, and main band (nuclear) DNA on a Hoechst/CsCl buoyant density gradient. The Pandorina mitochondrial DNA shows sufficient similarity to the 16 kb mitochondrial genome of Chlamydomonas reinhardtii to cross-hybridize, and also hybridizes with a probe containing maize mitochondrial 18S rRNA genes. Double digests, self-probing, and Bal31 exonuclease experiments suggest that 1.8 to 3.3 kb of sequence is repeated at each end of the genome as an inverted repeat. Mitochondrial genome sizes of other P. morum syngens were found to range from ca. 20 to ca. 38 kb. The mitochondrial genome should be valuable for taxonomic studies; it can be used for comparative organellar studies; and it should be of interest to compare with that of other plant and animal mitochondrial genomes.

Mitochondrial DNA variation in pearl millet and related species

Mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns and patterns of mtDNA hybridized by mitochondrial gene probes were used to study phylogenetic relationships of seven Pennisetum species, including five P. americanum (pearl millet) ecotypes and a reference species from the distantly related genus, Panicum. The restriction patterns of the pearl millet ecotypes were uniform with the exception of the ecotype collected in Ethiopia. The probe hybridization method revealed more variability, with both the Rhodesian and Ethiopian ecotypes differing from the others and from each other. Considerable restriction pattern polymorphism was noted among different species of Pennisetum, and Panicum. Significant relationships were noted of Pennisetum polystachyon to P. pedicellatum and of P. purpureum to P. squamulatum using the restriction pattern method. In addition to those relationships, the hybridization method showed relationships of pearl millet to P. purpureum and to P. squamulatum. The relationships noted between species by the hybridization method agreed more closely to the cytological data than those indicated by the restriction pattern method. Therefore, the hybridization method appeared to be the preferred method for studying species relationships. The mitochondrial genome size of pearl millet was calculated to be 407 kb and the mitochondrial genome sizes of other Pennisetum species ranged from 341 to 486 kb.

Species specificity of mitochondrial DNA flanking the 18S rRNA gene in two species of cucurbitaceae

Mitochondrial DNA is evolving slowly among species of Brassica and Lycopersicon. In contrast we have observed in cucurbits that at least 101 kilo bases (kb) out of 330 kb of watermelon mitochondrial DNA sequences are not found in muskmelon. The organization of the DNA flanking the mitochondrial 18S rRNA genes was compared in watermelon ( Citrullus vulgaris Schrad., cv. Dixie Queen) and muskmelon ( Cucumis melo L., cv. Iroquois), which differ 7-fold in mitochondrial genome size. Seventeen clones containing the 18S rRNA gene, averaging 14.9 ± 2.0 kb in size, were isolated from a lambda 1059 library of muskmelon mitochondrial DNA. The data are consistent with a single 18S rRNA gene in muskmelon mitochondrial DNA. Electron microscopy of heteroduplexes between watermelon and muskmelon clones containing the 18S rRNA gene show that the regions flanking the 18S gene in these two species are unrelated in nucleotide sequence. Southern blot analysis showed that the flanking sequences are unique to the respective species. At a reduced hybridization criterion these flanking sequences showed additional inter and intra species hybridization to both mitochondrial and chloroplast DNA indicating that the flanking regions contain either short reiterated sequences or longer sequences that have diverged greatly. Thus, in a region spanning 5% of the entire watermelon mitochondrial genome only a single highly conserved sequence, the 18S rRNA gene, is found.

Distribution of mitochondrial introns in the species Schizosaccharomyces pombe and the origin of the group II intron in the gene encoding apocytochrome b.

The mitochondrial genome size of 26 different Schizosaccharomyces pombe strains varies between 17.6 and 24.6 kilobase pairs due to the presence or absence of introns. One of these is the group II intron in the gene encoding apocytochrome b (cob: intron cobI1). Partial DNA sequences of continuous cob genes from six strains (including strain EF1: Trinkl et al. 1985) revealed identical nucleotide sequence in the region where the group II intron is inserted in the mosaic form of the gene. In contrast, analysis of the mosaic cob gene in strain UCD-FstI revealed several base pair changes in the exon regions flanking the splice point, compared with the continuous genes and with the mosaic cob gene in strain 50 (Lang et al. 1985). The base pair differences between the exons of the two mosaic cob genes and the identity of exons in all continuous cob genes argue in favour of the two cob introns in strains 50 and UCD-FstI as independent later acquisitions of the genes, rather than loss of the intron from a common mosaic ancestor of all strains. Other introns present in some but not all strain include two group I introns without open reading frame in the gene encoding subunit 1 of cytochrome c oxidase (cox1: introns cox1I2a and cox1I3), and two group I introns with open reading frames in the same gene (introns cox1I1 and cox1I2b).

Species delimitation in the Armillaria mellea complex by analysis of nuclear and mitochondrial DNAs

Nuclear and mitochondrial DNAs were isolated from mycelia of two different isolates of Armillaria mellea, A. bulbosa and A. obscura originating from widely separated localities. Nuclear GC contents ranged from 46.0 to 48.1 mol%. Nuclear DNA-DNA homologies obtained by a fast spectroscopic method were 90–100% for intraspecific hybridizations and 44–70% for interspecific hybridizations. Restriction enzyme cleavage patterns of mitochondrial DNAs using six different restriction enzymes yielded interspecific similarities from 0 to 50% and intraspecific similarities from 67 to 100%. Phylogenetic distances of the fungal species could not be deduced unequivocally from the mtDNA restriction data. Average mitochondrial genome sizes were estimated to be between approximately 90 and 100 kb.

Mitochondrial DNA Restriction site polymorphism in Drosophila montana and drosophila virilis

Mitochondrial DNA fragment patterns produced by ten restriction endonucleases have been studied for three geographically diverse Drosophila montana lines and two Drosophila virilis lines. The two species are estimated to differ at about 6% of mitochondrial DNA nucleotide sites. In both cases intraspecific genetic variation is relatively small (pairs of lines are estimated to differ at about 0.6% of mitochondrial DNA nucleotide sites). It appears that Japanese and North American D. montana populations have not been separated for an extended period. In D. virilis there is evidence for intraspecific variation in the size of the mitochondrial genome.

Large mitochondrial genome and mitochondrial DNA size polymorphism in the mosquito parasite, Romanomermis culicivorax.

Physical characterization of the mitochondrial genome derived from the obligate mosquito parasite, Romanomermis culicivorax has generated some surprising physical properties regarding the molecular structure of nematode mitochondrial DNA (mtDNA). Restriction enzyme analysis of this mtDNA has revealed a mitochondrial genome size of approximately 26 kb, the largest metazoan mtDNA reported to date. Isofemale lineages are monomorphic for one of three size variants, differing by 500-1,000 base pairs, present in our original field population. Cloned hybridization probes derived from a single region exhibiting a 600 bp size polymorphism share strong homology with several spatially separated sites distributed about the mtDNA. This suggests that the homology is a result of repeated DNA sequence elements contained within this mitochondrial genome that contribute to mtDNA size polymorphism.

Molecular analysis of mitochondrial DNA from tomato cell suspension cultures

Mitochondrial DNA (mtDNA) from Lycopersicon esculentum was purified from cell suspension cultures. The DNA, isolated from mitochondria purified by two successive sucrose density gradients, was uncontaminated with nuclear DNA or DNA from proplastids. The total molecular weights of BamHI, BglI, and BglII fragments indicate a mitochondrial genome size of at least 270 kb. Cross hybridization between tomato mtDNA and cloned spinach plastid genes revealed some homology. In hybridization experiments using cloned mitochondrial rRNA genes and BamHI digested total mtDNA the presence of recombination repeats is demonstrated.

Variation in Mitochondrial DNA Genome Size among Fishes of the Family Scorpaenidae

Variation in Mitochondrial DNA Genome Size among Fishes of the Family Scorpaenidae

Overlapping reading frames in Oenothera mitochondria

An open reading frame (ORF) preceding the cytochrome oxidase subunit II (CO II) gene in Oenothera mitochondria has four nucleotides in common with this gene. The last two nucleotides of the CO II initiation codon ATG are the first two nucleotides of the TGA termination codon in the upstream ORF. Both reading frames are cotranscribed in a bicistronic mRNA species of 1250 nucleotides in length in Oenothera. The open reading frame codes for a protein of 58 amino acids with structural homology to the ATPase subunit 8 genes in fungal and mammalian mitochondria. Using coding space optimally though overlapping genes appears to be without economical reason considering the large size of higher plant mitochondrial genomes.

Mitochondrial gene expression in Cucurbitaceae: conserved and variable features.

We have examined mitochondrial DNA (mtDNA) sequence conservation, transcriptional patterns of mitochondrial genes, and mitochondrial translation products in four species of the plant family Cucurbitaceae, in which there is a seven-fold range in mitochondrial genome size. A set of conserved mtDNA sequences which we term "core" DNA is present in all cucurbit genomes examined. In watermelon, only those mtDNA restriction fragments which contain "core" DNA hybridize with mtRNA. Similar numbers of polypeptides are synthesized by isolated mitochondria from all species examined, but an extra set of mtDNA sequences is transcribed in the largest genome (muskmelon). Taken together, these results suggest that some mtRNA is untranslated in muskmelon. Cloned mitochondrial genes of known function, from maize, identified mitochondrial transcripts that varied in both size and number among cucurbit species.

Isolation and physicochemical characterization of mitochondrial DNA from cultured cells ofPetunia hybrida

Mitochondrial DNA ofPetunia hybrida was purified from cell suspension cultures. Up to 50% of the DNA could be isolated as supercoiled DNA molecules by CsCl-ethidium bromide density gradient centrifugation. The DNA purified from DNase-treated mitochondria bands at a single buoyant density of 1.760 gcm(-3) in neutral density gradients and runs on agarose gels as a single band with an apparent molecular weight exceeding 30 megadaltons (Md). Summing of the restriction endonuclease fragment lengths indicates a mitochondrial genome size of at least 190 Md. Electron microscopic analysis reveals the presence of a heterogeneous population of circular DNA molecules, up to 60 Md in size. Small circular DNA molecules, ranging in size from 2-30 Md are present, but unlike in cultured cells of other plant species they do not form discrete size classes and furthermore, they constitute less than 5% of the total DNA content of the mitochondria. The restriction endonuclease patterns of mitochondrial DNA do not qualitatively alter upon prolonged culture periods (up to at least two years).