Homologies to plastid DNA in the nuclear and mitochondrial genomes of potato

Summary Potato plastid DNA clones, representing onefourth of the potato plastome complexity and containing sequences of the 16SrRNA, rps16, atpA, atpE, psaA, psaB, trnK, trnV, and trnG genes, were used as hybridization probes on nuclear- and mitochondrial-enriched DNAs. Each probe hybridized to multiple nuclear restriction fragments distinct from the plastid cleavage products generated by the same endonucleases. The nuclear hybridizable fragments are highly methylated at their Hpall target sequences (C/CGG). In some instances, the transfer seemed to involve plastid regions of several kilobase pairs, as reflected by the co-integration in the nucleus of restriction sites that are distant in the plastome. Three clones hybridized additionally to distinct mitochondrial fragments. These results indicate that extensive DNA transfers did occur between plastids and other organelles in potato.     

Sequencing complete mitochondrial and plastid genomes

Organelle genomics has become an increasingly important research field, with applications in molecular modeling, phylogeny, taxonomy, population genetics and biodiversity. Typically, research projects involve the determination and comparative analysis of complete mitochondrial and plastid genome sequences, either from closely related species or from a taxonomically broad range of organisms. Here, we describe two alternative organelle genome sequencing protocols. The "random genome sequencing" protocol is suited for the large majority of organelle genomes irrespective of their size. It involves DNA fragmentation by shearing (nebulization) and blunt-end cloning of the resulting fragments into pUC or BlueScript-type vectors. This protocol excels in randomness of clone libraries as well as in time and cost-effectiveness. The "long-PCR-based genome sequencing" protocol is specifically adapted for DNAs of low purity and quantity, and is particularly effective for small organelle genomes. Library construction by either protocol can be completed within 1 week.     

Gradients in plastid and mitochondrial DNA synthesis inFunaria protonemata: Visualization by bromodeoxyuridine immunohistochemistry

Summary DNA synthesis was investigated by visualizing sites of bromodeoxyuridine incorporation with antibodies in protonemata of the mossFunaria hygrometrica. In apically elongating tip cells a pronounced gradient of organelle DNA synthesis from tip to base was visible, reflecting the distribution of proliferating organelles within the tip cell. Side branch development coincided with reinitiation of replication of plastid and mitochondrial DNA.Abbreviations BrdU 5-bromo-2-deoxyuridine - DABCO diazabicyclol (2,2,2) octan - FITC fluorescein-5-isothiocyanat - HSA human serum albumine - IgG immunoglobulin G - PBS saline phosphate buffer     

Purification of mitochondrial DNA from total cellular DNA of small tissue samples

A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.     

Purification of plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata

We report a straightforward protocol for isolating plastid DNA from an enriched rhodoplast fraction of the red algaGracilaria tenuistipitata. Plastids were purified using differential centrifugation and 2-step sucrose density gradients. We found that 10% polyethylene glycol 4000 was essential for maintaining plastid integrity prior to lysis. Plastid DNA isolated directly from the purified rhodoplasts was sufficiently pure for restriction endonuclease fragment analyses. Database comparisons of sequences generated randomly from a shotgun genomic library indicated that plastid DNA was 89% pure following ultracentrifugation in isopycnic cesium chloride equilibrium gradients. The protocol yields 30–50 μg of plastid DNA per 100 g of fresh algal tissue.     

In situ plastid and mitochondrial DNA determination: Implication of the observed MINIMAL PLASTID GENOME NUMBER

The total loss of plastid DNA has never been reported for any alga or plant cell line, with the sole exception of the protozoan Euglena, yet plastid distribution at mitosis is apparently stochastric (Birky and Skavaril, Journal of Theoretical Biology, vol. 106, pp. 441–447, 1984) and accidental loss might be expected. It is not obvious how stem cells of photosynthetic eukaryotes avoid this problem. The chrysophyte alga Ochromonas danica, described as having but one or two plastids, can proliferate indefinitely without the benefit of photosynthesis. Under such conditions its plastid genome copy number per cell might drop to the absolute minimum compatible with maintaining its inheritance. In situ quantitation of Ochromonas plastid DNA in both photosynthetic and enriched mixotrophic growth, and in heterotrophic growth in prolonged darkness, suggests that plastids are capable of very wide variation (7 to >;200 genomes/plastid) in their DNA content, and likewise, cells can vary from one to >;8 plastids per cell, with total genomes numbers from 7 to >;1,000 per cell. Among many growth conditions tested, the smallest plastids were found in rapidly dividing cells grown in the dark, many of which contained but one plastid. The inability to find plastids with fewer than seven plastid genome equivalents of DNA, even in these rapidly multiplying cells grown in total darkness for months, suggests that multiple copies of the plastid genome may be very carefully maintained, even in the prolonged absence of photosynthesis. This implies that multiple copies are important for reasons other than photosynthetic capability; two possibilities are the biosynthetic steps necessary for eukaryote cell survival known to occur solely within a plastid, and/or the potential that multiple plastid genome copies provide to escape the effects of Muller's ratchet.     

Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development.

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.     

Purification of a mitochondrial DNA polymerase from Crithidia fasciculata.

The mitochondrial DNA polymerase from Crithidia fasciculata has been purified to near homogeneity. SDS-PAGE analysis of the purified enzyme reveals a single polypeptide with a molecular weight of approximately 43,000. The protein is basic, with an isoelectric point between 7.6-8.0. Its Stokes radius of 22 A and its sedimentation coefficient of 4.1 S suggest a native molecular weight of 38,000, indicating that the protein is a monomer under our experimental conditions. Western blots and immunoprecipitations of crude extracts reveal a cross-reacting protein of 48 kDa, suggesting that the purified enzyme may be an enzymatically active proteolytic product. The mitochondrial origin of the polymerase was confirmed by cell fractionation. Our results indicate that the C. fasciculata enzyme may be among the smallest known mitochondrial polymerases.     

Preferential degradation of plastid DNA with preservation of mitochondrial DNA in the sperm cells ofPelargonium zonale during pollen development

Summary In the present study, we studied changes in organellar DNA in the sperm cells of maturing pollen ofPelargonium zonale, a plant typical to exhibit biparental inheritance, by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and by immunogold electron microscopy using anti-DNA antibody. Fluorescence intensities of DAPI-stained plastid nuclei in generative and sperm cells at various developmental stages were quantified with a video-intensified microscope photon counting system (VIMPCS). Results indicated that the amount of DNA per plastid in generative cells increased gradually during pollen development and reached a maximum value (about 70 T per plastid; 1 T represents the amount of DNA in a particle of T4 phage) in young sperm cells at 5 days before flowering. However, the DNA content of plastids was subsequently reduced to about 20% of the maximum value on the day of flowering. Moreover, the DNA content of the plastid further decreased to 4% of the maximum value when pollen grains were cultured for 6 h in germination medium. In contrast, the amount of DNA per mitochondrion did not decrease significantly around the flowering day. Similar results were also obtained by immunogold electron microscopy using anti-DNA antibody. The density of gold particles on plastids decreased during pollen maturation whereas labelling density on mitochondria remained relatively constant. The number of plastids and mitochondria per generative cell or per pair of sperm cells did not change significantly, indicating that the segregation of DNA by plastid division was not responsible for the decrease in the amount of DNA per plastid. These results indicate that the plastid DNA is preferentially degraded, but the mitochondrial DNA is preserved, in the sperm cells ofP. zonale. While the plastid DNA of the sperm cells decreased before fertilization, it was also suggested that the low DNA contents that remain in the plastids of the sperm cells are enough to account for the biparental inheritance of plastids inP. zonale.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon counting system     

Interaction between plastid and mitochondrial retrograde signalling pathways during changes to plastid redox status

Mitochondria and chloroplasts depend upon each other; photosynthesis provides substrates for mitochondrial respiration and mitochondrial metabolism is essential for sustaining photosynthetic carbon assimilation. In addition, mitochondrial respiration protects photosynthesis against photoinhibition by dissipating excess redox equivalents from the chloroplasts. Genetic defects in mitochondrial function result in an excessive reduction and energization of the chloroplast. Thus, it is clear that the activities of mitochondria and plastids need to be coordinated, but the manner by which the organelles communicate to coordinate their activities is unknown. The regulator of alternative oxidase (rao1) mutant was isolated as a mutant unable to induce AOX1a expression in response to the inhibitor of the mitochondrial cytochrome c reductase (complex III), antimycin A. RAO1 encodes the nuclear localized cyclin-dependent kinase E1 (CDKE1). Interestingly, the rao1 mutant demonstrates a genome uncoupled phenotype also in response to redox changes in the photosynthetic electron transport chain. Thus, CDKE1 was shown to regulate both LIGHT HARVESTING COMPLEX B (LHCB) and ALTERNATIVE OXIDASE 1 (AOX1a) expression in response to retrograde signals. Our results suggest that CDKE1 is a central nuclear component integrating mitochondrial and plastid retrograde signals and plays a role in regulating energy metabolism during the response to stress.     

Purification and characterization of an inducible mitochondrial DNA polymerase from Tetrahymena thermophila

Treatment of the eukaryotic organism Tetrahymena with various types of DNA-damaging agents has been reported to cause a 35-fold induction of a mitochondrial DNA polymerase. We here report that the enzyme can be induced in large-scale cultures by exposure of the cells to thymine starvation and/or intercalating agents. The induced DNA polymerase has been purified to near homogeneity, with a specific activity of approx. 300,000 units/mg protein. The relative molecular mass of the active form of the enzyme is approx. 100,000, as determined by glycerol gradient sedimentation. The subunit structure has been analysed by SDS polyacrylamide gel electrophoresis of the highly purified preparation and by immunoprecipitation with a monoclonal antibody directed to the DNA polymerase. A polypeptide of Mr 47,000 has been observed to be a subunit of the enzyme. This corresponds to the size of the subunits suggested for mitochondrial DNA polymerase from chicken embryos and mouse myeloma cells.     

Arabidopsis calcium-binding mitochondrial carrier proteins as potential facilitators of mitochondrial ATP-import and plastid SAM-import

Chloroplasts and mitochondria are central to crucial cellular processes in plants and contribute to a whole range of metabolic pathways. The use of calcium ions as a secondary messenger in and around organelles is increasingly appreciated as an important mediator of plant cell signaling, enabling plants to develop or to acclimatize to changing environmental conditions. Here, we have studied the four calcium-dependent mitochondrial carriers that are encoded in the Arabidopsis genome. An unknown substrate carrier, which was previously found to localize to chloroplasts, is proposed to present a calcium-dependent S-adenosyl methionine carrier. For three predicted ATP/phosphate carriers, we present experimental evidence that they can function as mitochondrial ATP-importers.     

Purification and identification of subunit structure of the human mitochondrial DNA polymerase.

The mitochondrial DNA polymerase of HeLa cells was purified 18,000-fold to near homogeneity. The purified polymerase cofractionated with two polypeptides that had molecular mass of 140 and 54 kDa. The 140-kDa subunit was specifically radiolabeled in a photoaffinity cross-linking assay and is most likely the catalytic subunit of the mitochondrial DNA polymerase. The purified enzyme exhibited properties that have been attributed to DNA polymerase gamma and shows a preference for replicating primed poly(pyrimidine) DNA templates in the presence of 0.5 mM MgCl2. As in the case of mitochondrial DNA polymerases from other animal cells, human DNA polymerase gamma cofractionated with a 3'----5' exonuclease activity. However, it has not been possible to determine if the two enzymatic activities reside in the same polypeptide. The exonuclease activity preferentially removes mismatched nucleotides from the 3' end of a duplex DNA and is not active toward DNA with matched 3' ends. These properties are consistent with the notion that the exonuclease activity plays a proofreading function in the replication of the organelle genome.     

The foundation of extranuclear inheritance: plastid and mitochondrial genetics

In 1909 two papers by Correns and by Baur published in volume 1 of Zeitschrift für induktive Abstammungs- und Vererbungslehre (now Molecular Genetics and Genomics) reported on the non-Mendelian inheritance of chlorophyll deficiencies. These papers, reporting the very first cases of extranuclear inheritance, laid the foundation for a new field: non-Mendelian or extranuclear genetics. Correns observed a purely maternal inheritance (in Mirabilis), whereas Baur found a biparental inheritance (in Pelargonium). Correns suspected the non-Mendelian factors in the cytoplasm, while Baur believed that the plastids carry these extranuclear factors. In the following years, Baur’s hypothesis was proved to be correct. Baur subsequently developed the theory of plastid inheritance. In many genera the plastids are transmitted only uniparentally by the mother, while in a few genera there is a biparental plastid inheritance. Commonly there is random sorting of plastids during ontogenetic development. Renner and Schwemmle as well as geneticists in other countries added additional details to this theory. Pioneering studies on mitochondrial inheritance in yeast started in 1949 in the group of Ephrussi and Slonimski; respiration-deficient cells (petites in yeast, poky in Neurospora) were demonstrated to be due to mitochondrial mutations. Electron microscopical and biochemical studies (1962–1964) showed that plastids and mitochondria contain organelle-specific DNA molecules. These findings laid the molecular basis for the two branches of extranuclear inheritance: plastid and mitochondrial genetics.     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

Prokaryotic orthologues of mitochondrial alternative oxidase and plastid terminal oxidase

The mitochondrial alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) are two similar members of the membrane-bound diiron carboxylate group of proteins. AOX is a ubiquinol oxidase present in all higher plants, as well as some algae, fungi, and protists. It may serve to dampen reactive oxygen species generation by the respiratory electron transport chain. PTOX is a plastoquinol oxidase in plants and some algae. It is required in carotenoid biosynthesis and may represent the elusive oxidase in chlororespiration. Recently, prokaryotic orthologues of both AOX and PTOX proteins have appeared in sequence databases. These include PTOX orthologues present in four different cyanobacteria as well as an AOX orthologue in an alpha-proteobacterium. We used PCR, RT-PCR and northern analyses to confirm the presence and expression of the PTOX gene in Anabaena variabilis PCC 7120. An extensive phylogeny of newly found prokaryotic and eukaryotic AOX and PTOX proteins supports the idea that AOX and PTOX represent two distinct groups of proteins that diverged prior to the endosymbiotic events that gave rise to the eukaryotic organelles. Using multiple sequence alignment, we identified residues conserved in all AOX and PTOX proteins. We also provide a scheme to readily distinguish PTOX from AOX proteins based upon differences in amino acid sequence in motifs around the conserved iron-binding residues. Given the presence of PTOX in cyanobacteria, we suggest that this acronym now stand for plastoquinol terminal oxidase. Our results have implications for the photosynthetic and respiratory metabolism of these prokaryotes, as well as for the origin and evolution of eukaryotic AOX and PTOX proteins.