A replicating module as the unit of mitochondrial structure and functioning

The mitochondrion within human cells in tissue culture is pleomorphic and highly dynamic. The organelle mass can exist as thousands of small ovoids or as one continuous reticulum. In either state, the mitochondrial mass is in constant thermal motion, as well as moving in approximately 0.8-microm jumps that are determined by, and related to, attachments with cytoskeletal elements. Many protein complexes, such as the pyruvate dehydrogenase (PDH) complex and DNA containing nucleoids, are dispersed through the mass and as though fixed by attachments to membranes, such that they can become distributed to all of the individual small ovoid mitochondria when the reticulum becomes fragmented. This leads us to propose that a replicating module is the repeating unit of mitochondrial structure. Studies to examine heterogeneity of functioning within the organelle mass are briefly reviewed.     

Activation of human mitochondrial lysyl-tRNA synthetase upon maturation of its premitochondrial precursor

The cytoplasmic and mitochondrial species of human lysyl-tRNA synthetase are encoded by a single gene by means of alternative splicing of the KARS1 gene. The cytosolic enzyme possesses a eukaryote-specific N-terminal polypeptide extension that confers on the native enzyme potent tRNA binding properties required for the vectorial transfer of tRNA from the synthetase to elongation factor EF1A within the eukaryotic translation machinery. The mitochondrial enzyme matures from its precursor upon being targeted to that organelle. To understand how the cytosolic and mitochondrial enzymes are adapted to participate in two distinct translation machineries, of eukaryotic or bacterial origin, we characterized the mitochondrial LysRS species. Here we report that cleavage of the precursor of mitochondrial LysRS leads to a mature enzyme with reduced tRNA binding properties compared to those of the cytoplasmic counterpart. This adaptation mechanism may prevent inhibition of translation through sequestration of lysyl-tRNA on the synthetase in a compartment where the bacterial-like elongation factor EF-Tu could not assist in its dissociation from the synthetase. We also observed that the RxxxKRxxK tRNA-binding motif of mitochondrial LysRS is not functional in the precursor form of that enzyme and becomes operational after cleavage of the mitochondrial targeting sequence. The finding that maturation of the precursor is needed to reveal the potent tRNA binding properties of this enzyme has strong implications for the spatiotemporal regulation of its activities and is consistent with previous studies suggesting that the only LysRS species able to promote packaging of tRNA(Lys) into HIV-1 viral particles is the mature form of the mitochondrial enzyme.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Isolation of ATP2 coding the F1-ATPase beta subunit

A yeast nuclear pet mutant of Saccharomyces cerevisiae lacking any detectable mitochondrial F1-ATPase activity was genetically complemented upon transformation with a pool of wild type genomic DNA fragments carried in the yeast Escherchia coli shuttle vector YEp 13. Plasmid-dependent complementation restored both growth of the pet mutant on a nonfermentable carbon source as well as functional mitochondrial ATPase activity. Characterization of the complementing plasmid by plasmid deletion analysis indicated that the complementing gene was contained on adjoining BamH1 fragments with a combined length of 3.05 kilobases. Gel analysis of the product of this DNA by in vitro translation in a rabbit reticulocyte lysate programmed with yeast mRNA hybrid selected by the plasmid revealed a product which could be immunoprecipitated by antisera against the beta subunit of the yeast mitochondrial ATPase complex. A comparison of the protein sequence derived from partial DNA sequence analysis indicated that the beta subunit of the yeast mitochondrial ATPase complex exhibits greater than 70% conservation of protein sequence when compared to the same subunit from the ATPase of E. coli, beef heart, and chloroplast. The gene coding the beta subunit (subunit 2) of yeast mitochondrial adenosine triphosphatase is designated ATP2. The utilization of cloned nuclear structural genes of mitochondrial proteins for the analysis of the post-translational targeting and import events in organelle assembly is discussed.     

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid

Summary A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5 ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3 ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila

The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.     

Mitochondrial plasmids in related cultivars of crookneck and stable and unstable butternut squash

Summary Two series of two to four plasmids are contained in mitochondria of butternut squash (Cucurbita moschatd). The plasmids are composed of DNA, and are consistent in number within a cultivar but vary in number among cultivars. Plasmid patterns and homology suggest that the faster migrating series of plasmids are the supercoiled form of the slower running plasmids. Plasmid 1 was present in all cultivars except New Hampshire Butternut. Plasmid 2 was only observed in the original crookneck cultivar, Canada Crookneck. Plasmids 3 and 4 were present in all accessions tested. The presence of plasmid 1 in a cultivar does not follow a maternal pattern of inheritance; this may be due to the ability of the plasmid to insert into and excise from the main mitochondrial DNA, or the recombination of some of the smaller plasmids to create the larger plasmid 1. There is some homology between plasmid 1 and plasmids 3 and 4, and between plasmid 1 and main band mitochondrial DNA. The equal presence of plasmid 1 in mitochondria of F₁ seedlings from the pollination of New Hampshire Butternut by Ponca Butternut to that in F₁ seedlings of the reciprocal cross indicates that there is probably a dominant nuclear effect on the ability to produce plasmid 1 either by release from the mitochondrial genome or by recombination of the smaller plasmids. There is no obvious relationship between the presence or number of the mitochondrial plasmids and the butternut fruit shape or stability of the butternut trait of the cultivars.     

Mitochondrial genetics and disease

Mitochondrial respiratory chain diseases are a highly diverse group of disorders whose main unifying characteristic is the impairment of mitochondrial function. As befits an organelle containing gene products encoded by both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), these diseases can be caused by inherited errors in either genome, but a surprising number are sporadic, and a few are even caused by environmental factors.     

Divergent potentials for cytoplasmic inheritance within the genus Syringa. A new trait associated with speciogenesis

Epifluorescence microscopic detection of organelle DNA in the mature generative cell is a rapid method for determining the potential for the mode of cytoplasmic inheritance. We used this method to examine 19 of the known 22 to 27 species in the genus Syringa. Organelle DNA was undetectable in seven species, all in the subgenus Syringa, but was detected in the 12 species examined of the subgenera Syringa and Ligustrina. Therefore, species within the genus Syringa display differences in the potential cytoplasmic inheritance. Closer examination revealed that the mature generative cells of the species in which organelle DNA was detected contained both mitochondria and plastids, but cells of the species lacking detectable organelle DNA contained only mitochondria, and the epifluorescent organelle DNA signals from the mature generative cells corresponded to plastid DNA. In addition, semiquantitative analysis was used to demonstrate that, during pollen development, the amount of mitochondrial DNA decreased greatly in the generative cells of the species examined, but the amount of plastid DNA increased remarkably in the species containing plastids in the generative cell. The results suggest that all Syringa species exhibit potential maternal mitochondrial inheritance, and a number of the species exhibit potential biparental plastid inheritance. The difference between the modes of potential plastid inheritance among the species suggests different phylogenies for the species; it also supports recent conclusions of molecular, systematic studies of the Syringa. In addition, the results provide new evidence for the mechanisms of maternal mitochondrial inheritance in angiosperms.     

RAPD markers of mitochondrial origin exhibit lower population diversity and higher differentiation than RAPDs of nuclear origin in Douglas fir

We developed a method of screening RAPD markers for the presence of organelle DNA products using enriched organelle DNA probes, then used these markers to compare the structure of nuclear and mitochondrial RAPD diversity in Douglas fir. Of 237 screened RAPD fragments from 25 primers, 16% were identified as originating in the mitochondrial genome and 3% in the chloroplast genome. The mitochondrial DNA probe correctly distinguished fragments with known maternal inheritance (which is exclusive for the mitochondrial genome in the Pinaceae), and neither of the organelle probes hybridized to biparentally inherited fragments. Mitochondrial RAPD markers exhibited low diversity within populations compared to nuclear RAPD diversity ( H _S = 0.03 and 0.22, respectively), but were much more highly differentiated than were fragments of nuclear origin at both the population ( G _ST = 0.18 and 0.05, respectively) and racial levels ( G _ST = 0.72 and 0.25, respectively). Both nuclear and mitochondrial DNA based phylogenetic analyses identified the varieties as monophyletic groups; the nuclear RAPD markers further separated the north and south interior races.     

Purification of mitochondrial and plastid DNA

The size, structure and conformation of mitochondrial and plastid genomes differ dramatically among eukaryotes. Similarly, the yield and purity of extracted organelle DNA also vary, and are crucial factors for the success of restriction mapping and sequencing experiments. We describe here procedures for the purification of organelle DNA from a broad range of eukaryotes. By emphasizing the underlying principles, these procedures will facilitate the development of new species-specific protocols. The presented purification schemes involve either isolation of organelles and subsequent extraction of DNA from this subcellular fraction, or processing of whole-cell lysates followed by CsCl gradient centrifugation to separate nuclear and organelle DNAs according to their A + T content. We have successfully used the described procedures for organelle genome sequencing from diverse eukaryotes, including non-axenic protists. Procedures can be completed in 3-5 days, typically yielding a few micrograms of DNA-ample for sequencing complete genomes.     

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.     

Linear kalilo DNA is a Neurospora mitochondrial plasmid that integrates into the mitochondrial DNA

Summary The linear autonomous form of kalilo DNA (previously called AR-kalDNA) is shown to be resident within mitochondria rather than nuclei, as had been suggested by previous experiments. This form has been renamed mtAR-kalDNA, to signify its mitochondrial location. Experiments are described that illustrate the inheritance and somatic transmission patterns of the mitochondrial kalilo plasmid and the mitochondrial inserted form of kalilo DNA (mtlS-kalDNA). Progeny of a cross with a pre-senescent subculture as the female parent inherited mtAR-ka1DNA only; mtIS-kalDNA was not transmitted sexually. During somatic propagation of the ascospore cultures, novel kalilo DNA inserts appeared and most of them persisted until death. We propose that these inserts originated from de novo integration of mtAR-kalDNA into the mitochondrial DNA. In two of the ascopore-derived series analyzed, the first inserts detected were seen only transiently and inserts appearing subsequent to the transient inserts were retained until death. We propose that these enduring inserts originated either from rearrangements of the transient inserts or from novel integration events, either from mtAR-kalDNA or from transposition of the transient inserts.     

Plastid DNA diversity in natural populations of Beta maritima showing additional variation in sexual phenotype and mitochondrial DNA

Summary Plants of two natural populations of Beta maritima, characterized by high percentages of male-sterile plants, have been investigated for organelle DNA polymorphism. We confirm the two classes of mitochondrial DNA variation previously described: (i) mitochondrial DNA (mtDNA) type N is associated with male fertility, whereas mtDNA type S can cause cytoplasmic male sterility (CMS); (ii) the 10.4-kb linear plasmid is observed in both types of mitochondria and is not correlated with the cytoplasmic male sterility occurring in this plant material. A third polymorphism is now described for chloroplast DNA (ctDNA). This polymorphism occurs within single populations of Beta maritima. Three different ctDNA types have been identified by HindIII restriction analysis. Among the plants studied, ctDNA type 1 is associated with N mitochondria and type 2 with S mitochondria. Chloroplast DNA type 3 has been found both in a fertile N plant and in a sterile S plant. This finding suggests that the chloroplast DNA polymorphism reported is not involved in the expression of male sterility. A comparison with Beta vulgaris indicates that ctDNA type 3 of Beta maritima corresponds to the ctDNA of fertile sugar beet maintainer lines. The three types of Beta maritima ctDNA described in this study differ from the ctDNA of male-sterile sugar beet.     

Sequences required for delivery and localization of the ADP/ATP translocator to the mitochondrial inner membrane.

The ADP/ATP translocator, a transmembrane protein of the mitochondrial inner membrane, is coded in Saccharomyces cerevisiae by the nuclear gene PET9. DNA sequence analysis of the PET9 gene showed that it encoded a protein of 309 amino acids which exhibited a high degree of homology with mitochondrial translocator proteins from other sources. This mitochondrial precursor, in contrast to many others, does not contain a transient presequence which has been shown to direct the posttranslational localization of proteins in the organelle. Gene fusions between the PET9 gene and the gene encoding beta-galactosidase (lacZ) were constructed to define the location of sequences necessary for the mitochondrial delivery of the ADP/ATP translocator protein in vivo. These studies reveal that the information to target the hybrid molecule to the mitochondria is present within the first 115 residues of the protein. In addition, these studies suggest that the "import information" of the amino-terminal region of the ADP/ATP translocator precursor is twofold. In addition to providing targeting function of the precursor to the organelle, these amino-terminal sequences act to prevent membrane-anchoring sequences located between residues 78 and 98 from stopping import at the outer mitochondrial membrane. These results are discussed in light of the function of distinct protein elements at the amino terminus of mitochondrially destined precursors in both organelle delivery and correct membrane localization.     

Inheritance of chloroplast and mitochondrial genomes in pedunculate oak investigated with an efficient PCR method

The restriction patterns of two chloroplast fragments and one mitochondrial DNA fragment, amplified by PCR with universal primers, were studied to determine the mode of inheritance of these organelles in 143 progeny of five intraspecific crosses in pedunculate oak (Quercus robur L.). The results indicate that both genomes are maternally inherited, an observation which agrees with the commonly observed pattern of inheritance in angiosperms. They confirm that both chloroplast DNA and mitochondrial DNA can be used as a source of seed-specific markers for the study of the geographic structure of oaks. This is the first report of organelle inheritance within the Fagaceae, an important and widespread tree family.     

Micro Review Bending the rules: the 2μ plasmid of yeast

The replication of euckaryotic DNA is normally initiated at each origin only once per ceil cycle. Yet, In spite of this restriction, the 2μ plasmid of yeast has evolved an elegant mechanism which can allow it to rapidly amplify its copy number without initiating multiple rounds of replication. It achieves this by exploiting a plasmid-en coded site-specific recombination system in a way that is apparently unique to this plasmid. The 2μ plasmid has also evolved a mechanism that allows effective partition of itself between mother and daughter cells. Together these processes ensure the persistence of the 2μ plasmid within a population, even though retention of the plasmid Is of no advantage to the host cell and causes a slightly slower growth rate. The success of this survival strategy is illustrated by the near ubiquity of the 2μ. plasmid in both wild-type and laboratory strains of yeast.