Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. I. Ultrastructural analysis of organelles

Electron microscope studies have been made on the fine structure of the colorless biflagellate, Polytoma obtusum, with main emphasis on the structural organization of the mitochondria and the leucoplast. Both organelles have been demonstrated to contain DNA aggregates as well as ribosomal particles within their matrix material. Reconstructions from serial showed that (a) the mitochondria were highly convoluted and irregular in shape and size, and (b) the leucoplast was a single cup-shaped entity, with large starch grains, localized at the posterior end, and multiple sites of DNA aggregates. The starch-containing compartments appeared to be interconnected by narrow tubular or sheetlike bridges. Cytoplasmic invaginations into the plastid, often containing mitochondria, were of frequent occurrence, and membranes of mitochondria and the leucoplast appeared to be closely apposed. Membranes elements, both sheetlike and vesicular, were also present in the matrix. The Polytoma leucoplast was, in certain respects, morphologically similar to the plastids of various photosynthetic mutants of Chlamydomonas, most of which show Menedelian segregation. It is suggested that Polytoma arose from a Chlamydomonas-like ancestor, possibly through combined mutational processess of both chloroplast and nuclear genomes. Since Polytoma leucoplasts contain both DNA and ribosomal particles, it is probable that these organelles still possess semiautonomy and limited ability for protein synthesis.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. II. General characterization of organelle nucleic acids.

Polytoma obtusum has a main band DNA (alpha) with a buoyant density in CsC1 of rho = 1.711 g/ml and a light DNA satellite (beta) with rho = 1.682 g/ml. beta-DNA was substantially enriched in a fraction containing small leucoplast fragments and some mitochondria, which was obtained in a pellet sedimenting between 3,000 g and 5,000 g. A crude mitochondrial pellet was also obtained by sedimenting at 12,000 g to recover particulates remaining in the supernate after 10 min at 5,000 g. This fraction contained a third DNA component (gamma) with rho = 1.714 g/ml. We have concluded that the leucoplasts of P. obtusum contain the beta-DNA (1.6882) and the mitochondria possess the gamma-component (1.714). Two distinct classess of ribosomes were isolated and separated by sucrose density gradients, a major 79S species and a minor species at 75S. The major species possessed the 25S and 18S ribosomal RNA (rRNA), characteristic of cytoplasmic ribosomes, and these particles co-sedimented in sucrose gradients with the 79S cytoplasmic ribosomes of Chlamydomonas reinhardtii. The minor species was present in about 2% of the total ribosomal population but showed an eight-to-ninefold enrichment in the leucoplast pellet, suggesting that it was of organelle origin. These 73S particles had RNA components migrating very closely with the 18S and 25S species of the 79S ribosomes, but the base composition of the rRNA from these two classes of ribosomes was significantly different; the rRNA from the 79S ribosomes had a G+C mole ratio of 50.0%, while the rRNA from the 73S class had a ratio of 47.5%. By comparison, chloroplast ribosomes of C. reinhardtii were found to sediment at 70S and contain rRNA molecules of 23S and 16S, with a G + C content of 51.0%. These findings support the concept that the Polytoma leucoplast possesses characteristic genetic and protein-forming systems.     

Characterization of cytoplasmic and nuclear genomes in the colorless alga Polytoma. III. Ribosomal RNA cistrons of the nucleus and leucoplast

The colorless alga Polytoma obtusum has been found to possess leucoplasts, and two kinds of ribosomes with sedimentation values of 73S and 79S. The ribosomal RNA (rRNA) of the 73S but not the 79S ribosomes was shown to hybridize with the leucoplast DNA (rho - 1.682 g/ml). Nuclear DNA of Polytoma (rho = 1.711) showed specific hybridization with rRNA from the 79S ribosomes. Saturation hybridization indicated that only one copy of the rRNA cistrons was present per leucoplast genome, with an average buoyant density of rho = 1.700. On the other hand, about 750 copies of the cytoplasmic rRNA cistrons were present per nuclear genome with a density of rho = 1.709. Heterologous hybridization studies with Chlamydomonas reinhardtii rRNAs showed an estimated 80% homology between the two cytoplasmic rRNAs, but only a 50% homology between chloroplast and leucoplast rRNAs of the two species. We conclude that the leucoplasts of Polytoma derive from chloroplasts of a Chlamydomonas-like ancestor, but that the leucoplast rRNA cistrons have diverged in evolution more extensively than the cistrons for cytoplasmic rRNA.     

Comparative investigation of the cytoplasmic ribosomal proteins of Chlamydomonas reinhardii and Polytoma mirum

The cytoplasmic and ribosomal proteins of Chlamydomonas reinhardii and Polytoma mirum were compared by 2-D gel electrophoresis. 56 proteins with molecular weights from 12 500 to 46 000 could be detected in Chlamydomonas and 53 proteins with molecular weights from 10 000 to 50 000 in Polytoma. 15 ribosomal proteins from Chlamydomonas were identical with those of Polytoma, and it could be concluded that 25 were corresponding to each other.     

Regeneration of somatic hybrids in relation to the nuclear and cytoplasmic genomes of wheat and Setaria italica.

Somatic hybridization via PEG (Polyethylene 6000)-mediated protoplast fusion was achieved between two different wheat culture lines (Triticum aestivum L., "Jinan"177, T1 and T2) and Setaria italica (L.) P. Beauv. The T1 recipient originated from non-regenerable long-term cell suspensions, while T2 was derived from embryogenic calli with a high regeneration capacity. Donor protoplasts were obtained from embryogenic calli of S. italica (S) (with low regeneration capacity) irradiated with different doses of ultraviolet light. Twenty-three putative hybrid cell lines were produced in fusion combinations with the donor protoplasts treated with UV light for 30 s (combination I) and 1 min (combination II), but only one (from combination II) differentiated into green plants. Three cell lines from combination I and five cell lines from combination II possessed the nuclear genomes of T1, T2, and S. italica as revealed by cytological, isozyme, RAPD, and 5S rDNA spacer sequence analyses. Genomic in situ hybridization (GISH) analysis showed that most hybrid cell lines had 22-36 wheat chromosomes, 0-2 S. italica chromosomes, and 1-6 wheat - S. italica recombinant chromosomes, whereas the regenerable cell line had 44-56 wheat chromosomes and 3-6 recombinant chromosomes, but no intact S. italica chromosomes. RFLP analysis of organellar DNA revealed that mitochondrial and chloroplast DNA of both parents coexisted in all hybrid cell lines and recombined in most hybrid cell lines. These results indicate that the regeneration of hybrid plants involves not only the integration of S. italica nuclear and organellar DNA, but also the genome complementation of T1 and T2.     

On the adaptive value of cytoplasmic genomes in plants

Is DNA variation maintained in organelle genomes selectively neutral? The answer to this question has important implications for many aspects of ecology and evolution. While traditionally the answer has been ‘yes’, recent studies in animals have shown that, on the contrary, mitochondrial DNA polymorphism is frequently adaptive. In plants, however, the neutrality assumption has not been strongly challenged. Here, we begin with a critical evaluation of arguments in favour of this long‐held view. We then discuss the latest empirical evidence for the opposing prediction that sequence variation in plant cytoplasmic genomes is frequently adaptive. While outstanding research progress is being made towards understanding this fundamental topic, we highlight the need for studies that combine information ranging from field experiments to physiology to molecular evolutionary biology. Such an interdisciplinary approach provides a means for determining the frequency, drivers and evolutionary significance of adaptive organelle DNA variation.     

Interactions between cytoplasmic and nuclear genomes confer sex‐specific effects on lifespan in Drosophila melanogaster

Genetic variation outside of the cell nucleus can affect the phenotype. The cytoplasm is home to the mitochondria, and in arthropods often hosts intracellular bacteria such as Wolbachia. Although numerous studies have implicated epistatic interactions between cytoplasmic and nuclear genetic variation as mediators of phenotypic expression, two questions remain. Firstly, it remains unclear whether outcomes of cyto‐nuclear interactions will manifest differently across the sexes, as might be predicted given that cytoplasmic genomes are screened by natural selection only through females as a consequence of their maternal inheritance. Secondly, the relative contribution of mitochondrial genetic variation to other cytoplasmic sources of variation, such as Wolbachia infection, in shaping phenotypic outcomes of cyto‐nuclear interactions remains unknown. Here, we address these questions, creating a fully crossed set of replicated cyto‐nuclear populations derived from three geographically distinct populations of Drosophila melanogaster, measuring the lifespan of males and females from each population. We observed that cyto‐nuclear interactions shape lifespan and that the outcomes of these interactions differ across the sexes. Yet, we found no evidence that placing the cytoplasms from one population alongside the nuclear background of others (generating putative cyto‐nuclear mismatches) leads to decreased lifespan in either sex. Although it was difficult to partition mitochondrial from Wolbachia effects, our results suggest at least some of the cytoplasmic genotypic contribution to lifespan was directly mediated by an effect of sequence variation in the mtDNA. Future work should explore the degree to which cyto‐nuclear interactions result in sex differences in the expression of other components of organismal life history.     

Oxidative phosphorylation supercomplexes and respirasome reconstitution of the colorless alga Polytomella sp.

The proposal that the respiratory complexes can associate with each other in larger structures named supercomplexes (SC) is generally accepted. In the last decades most of the data about this association came from studies in yeasts, mammals and plants, and information is scarce in other lineages. Here we studied the supramolecular association of the F₁F_O-ATP synthase (complex V) and the respiratory complexes I, III and IV of the colorless alga Polytomella sp. with an approach that involves solubilization using mild detergents, n-dodecyl-β-D-maltoside (DDM) or digitonin, followed by separation of native protein complexes by electrophoresis (BN-PAGE), after which we identified oligomeric forms of complex V (mainly V₂ and V₄) and different respiratory supercomplexes (I/IV₆, I/III₄, I/IV). In addition, purification/reconstitution of the supercomplexes by anion exchange chromatography was also performed. The data show that these complexes have the ability to strongly associate with each other and form DDM-stable macromolecular structures. The stable V₄ ATPase oligomer was observed by electron-microscopy and the association of the respiratory complexes in the so-called “respirasome” was able to perform in-vitro oxygen consumption.     

Characterization of p80, a novel nuclear and cytoplasmic protein in dinoflagellates.

The presence of myosin in dinoflagellates was tested using an anti-Acanthamoeba castellanii myosin II polyclonal antibody on the heterotrophic dinoflagellate Crypthecodinium cohnii Seligo. Western blots revealed the presence of a unique band of 80 kDa in total protein extracts and after immunoprecipitation. Expression of this 80 kDa protein appeared constant during the different phases of the cell cycle. In protein extracts from various other dinoflagellates, this 80 kDa protein was detected only in the autotrophic species Prorocentrum micans Ehr. Screening of a C. cohnii cDNA expression library with this antibody revealed a cDNA coding for an amino acid sequence without homology in the databases. However, particular regions were detected: - a polyglutamine repeat domain in the N-terminal part of the protein, - four peptide sequences associated with GTP-binding sites, - a sequence with slight homology to the rod tail of Caenorhabditis elegans myosin II, -a sequence with homology to a human kinesin motor domain. Immunocytolocalization performed on C. cohnii thin sections with a polyclonal antibody raised against the recombinant protein showed p80 to be present both within the nucleus and in the cytoplasm. Labelling was widespread in the nucleoplasm and more concentrated at the periphery of the permanently condensed chromosomes. In the cytoplasm, labelling appeared in a punctate region close to the nucleus and in the flagellum. Potential functions of this novel protein are discussed.     

Characterization of genetic diversity of nuclear and mitochondrial genomes in Daucus varieties by RAPD and AFLP

The genetic diversity of nuclear genomes of five Daucus species and seven Daucus carota L. subspecies involving 26 accessions was characterized with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). AFLP produced more than four times as many discrete bands per reaction compared with RAPD analysis, while both AFLP and RAPD basically led to similar conclusions. The dendrograms constructed with both RAPD and AFLP revealed that all accessions of D. carota were grouped into a major cluster delimited from other Daucus species, in good agreement with the classification by morphological char-acteristics. All accessions of cultivated carrots [(D. carota ssp. sativus (Hoffm.) Arcang.] were clustered in the same group while the variation within D. carota was relatively extensive. Genetic diversity of mitochondrial genomes was also documented with RAPD for the same accessions. The mitochondrial dendrogram differed from that of the nuclear genome, suggesting that nuclear and mitochondrial genomes of some accessions had separate evolutionary histories. Received: 20 September 1997 / Revision received: 19 January 1998 / Accepted: 28 March 1998     

Characterization of DNA components from some colorless algae

The DNA components of five colorless algae were characterized by their buoyant densities in cesium chloride. Two DNA components were detected in Polytoma obtusum and Polytoma uvella. Upon renaturation of the thermally denatured DNA the minor and approx. 15% of the major DNA component returned to their native densities. The buoyant densities of the major and minor DNA of P. obtusum and P. uvella are different from that of the morphologically and biochemically similar green alga Chlamydomonas reinhardtii. A major and a minor DNA component with the same buoyant densities as that of the green alga Euglena were also found in Astasia longa, which is morphologically similar to Euglena. The renaturation of the minor but not the major component was readily detectable by the change in buoyant density. Only one DNA component was detected in Polytomella agilis and Polytomella caeca. After thermal denaturation approx. 5% of each of these DNA components were renatured readily. Based on these data, the possible evolutionary origin of these colorless algae is discussed.     

Mitochondrial pseudogenes in the nuclear genomes of Drosophila

Mitochondrial pseudogenes in nuclear chromosomes (numts) have been detected in the genomes of a diverse range of eukaryotic species. However, the numt content of different genomes and their properties is not uniform, and study of these differences provides insight into the mechanisms and dynamics of genome evolution in different organisms. In the genus Drosophila, numts have previously only been identified on a genome-wide scale in the melanogaster subgroup. The present study extends the identification to 11 species of the Drosophila genus. We identify a total of 302 numts and show that the numt complement is highly variable in Drosophilids, ranging from just 4 in D. melanogaster to 67 in D. willistoni, broadly correlating with genome size. Many numts have undergone large-scale rearrangements in the nucleus, including interruptions, inversions, deletions and duplications of sequence of variable size. Estimating the age of the numts in the nucleus by phylogenetic tree reconstruction reveals the vast majority of numts to be recent gains, 90% having arisen on terminal branches of the species tree. By identifying paralogs and counting duplications among the extant numts we estimate that 23% of extant numts arose through post-insertion duplications. We estimate genus average rates of insertion of 0.75 per million years, and a duplication rate of 0.010 duplications per numt per million years.     

Biolistic co-transformation of the nuclear and plastid genomes

Particle gun-mediated (so-called 'biolistic') transformation represents a universal genetic transformation technology that is widely applied in nearly all groups of organisms. The mechanism of how accelerated DNA-coated particles, after their entry into the cell, deliver the foreign DNA to the target compartment is not known. Here we have studied this process in plants by performing co-transformation experiments with vectors targeted to two different cellular compartments, the nucleus and the plastids (chloroplasts). We find that coating of particles with both plastid and nuclear transformation vectors can result in co-transformation of chloroplasts and the nucleus. In contrast, mixing of particles coated individually with the vectors does not produce co-transformed plants. Our data suggest that a single DNA-coated particle can transform more than one compartment of the plant cell, opening up the possibility to generate doubly transgenic plants in one step. Importantly, co-transformation can also be obtained in the absence of selection, thus providing a method to produce marker-free transgenic genomes. In addition, our findings raise the possibility of occasional inadvertent co-transformation of two genomes and, therefore, have important implications for the molecular characterization and regulation of transgenic plants.     

Molecular characterization of the nuclear and cytoplasmic genomes of intergeneric diploid plants from cell fusion between Microcitrus papuana and Rough lemon

Simple-sequence-repeat (SSR) and PCR-RFLP were employed to characterize the nuclear and cytoplasmic genomes of intergeneric diploid plants derived from symmetric fusion between Microcitrus papuana Swingle and Rough lemon ( Citrus jambhiri Lush). Three out of five SSR primers distinguished the fusion parents from each other and the regenerated plants showed band profiles completely identical to the leaf parent, Rough lemon. Amplified products from the intergenic regions of cpDNA between trnD -trnT were digested with HaeIII and MspI, and those between trnH -trnK were digested with HinfI, and both the regenerated plants and Rough lemon shared the same band patterns, which were different from the embryogenic parent, M. papuana. With mtDNA, only 2 out of 12 primer pair/restriction enzyme combinations ( nad4 ex 1–2/ TaqI and nad4 ex 1–2/ HindIII) revealed polymorphisms between the fusion parents. With the former combination the regenerated plants showed the same fragment distribution as that of the embryogenic parent, M. papuana, whereas with the latter, a novel band absent in the fusion parents was detected in all of the regenerated plants, suggesting a possible rearrangement. The present research indicates that the plants analyzed were putative cybrids containing nuclear DNA and cpDNA from Rough lemon and mtDNA from M. papuana. Presumed mechanisms leading to the regeneration of diploid hybrid plants following symmetric fusion are discussed herein.