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  • 1
    ISSN: 1615-6102
    Keywords: Maternal inheritance ; Bryopis maxima ; Anisogamous alga ; Gametogenesis ; Preferential digestion ; DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1615-6102
    Keywords: Chloroplast division ; Unicellular red alga ; Cyanidium caldarium ; DNA content
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The behavior and DNA content of the cell and chloroplast nuclei (synonymous with nucleoids; ct-nuclei) during the life cycle have been studied in a synchronized population of cells of the unicellular algaCyanidium caldarium M-8. Cells were examined by epifluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI), and by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS). The young cell contains a single petal-like chloroplast, a spherical cell nucleus and several mitochondria, and the cell nucleus and the chloroplast divide in that order just prior to cytokinesis. The chloroplast contains a ring-shaped ct-nucleus which is located at the periphery of the chloroplast during the life cycle. During the first 40 h after the initiation of sychronous cultures, the young cell and its chloroplast increase markedly in size, and the DNA contents per cell nucleus and per ct-nucleus increase approximately two times and 16 times the value in 16-endospore cells, respectively. Four endospore divisions then occur, at intervals of approximately 12 h between 40 h and 90 h, after the initiation of synchronous cultures. The volume of each cell, the volume of each chloroplast, the amount of chloroplast DNA (ct-DNA), and the level of pigmented material in the chloroplast are reduced stepwise after each endospore division until finally, at the 16-endospore stage, they reach approximately 1/16 of the original values for the mother cell. The size of the mitotic spindle also is reduced stepwise as the cell divisions proceed. By contrast, the cell nuclei duplicate their DNA during each endospore division cycle. These results and analysis of other components indicate that the chloroplasts divide into two daughter chloroplasts without any DNA synthesis during four successive cycles of endospore division and also that the DNA content of the chloroplast is intimately related to the volume of the chloroplast and the cell and to the level of pigmented material in the cloroplast, but is not related to the DNA content of the cell nuclei.
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  • 3
    ISSN: 1615-6102
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1615-6102
    Keywords: Maternal inheritance ; Bryopsis maxima ; Anisogamous alga ; Gametogenesis ; Differential digestion ; Organelle nuclei (nucleoids)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The fate of chloroplast nuclei (cp-nuclei) and mitochondrial nuclei (mt-nuclei) was followed during gametogenesis in male and female coenocytic thalli in the anisogamous algaBryopsis maxima by epifluorescence microscopy, after staining with 4′,6-diamidino-2-phenylindole (DAPI), by quantification of chloroplast DNA (cp-DNA) by fluorimetry using a video-intensified, photon-counting system (VIMPICS), and by CsCl density gradient centrifugation. The male and female coenocytic thalli, 48 h before the release of gametes, contain a large number of chloroplasts, each of which is larger in size than the cell nucleus and the mitochondria and contains about 150 cp-nuclei. The size of each chloroplast in the female and male gametangia decreases markedly during gametogenesis as a result of continuous divisions till about 10 h before the release of gametes and, eventually, the numbers of cp-nuclei per chloroplast in the male and female gametangia fall to about 20 and 5, respectively. Two hours later, as the preferential digestion of cp-DNA in the male gametangium occurs, the number of cp-nuclei in the chloroplast of each male gamete falls to zero while the number of cp-nuclei in female gamete does not change, even after release of female gametes. Several mt-nuclei are observed in all of the female gametes. By contrast, the mt-nuclei in the bulk of the male gametes disappear but those in a few gametes remain. The profiles after CsCl density gradient centrifugation of DNAs extracted from male and female plants and gametes support the cytological data. The results suggest that the preferential digestion of cp-DNA in male plants occurs about 8 h before the release of gametes and that there is differential digestion of cp-DNA and mitochondrial DNA (mt-DNA).
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  • 5
    ISSN: 1615-6102
    Keywords: Physarum polycephalum ; DAPI ; Fluorescence microscopy ; Centrosome ; Comigration ; Centrosome migration ; Cell-nuclear migration ; Actin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The mechanism of cell-nuclear migration during the amoebo-flagellate transformation inPhysarum polycephalum was examined by fluorescence microscopy after staining with a tubulinspecific antibody, rhodamine-conjugated phalloidin and 4′,6-diamidino-2-phenylindole (DAPI). While the round amoeba cells changed to comma-shaped swarm cells within 20min after suspension in buffer, the cell nuclei moved from the central region of each cell to the periphery, each forming a sharp projection in the direction of movement. A centrosome also migrated from the center of the cell to the cell periphery. Since the centrosome was in close contact with the tip that protruded from the cell nucleus throughout the cellnuclear migration, the migration of the cell nucleus and the centrosome could be recognized as comigration. Then the flagella began to elongate from the centrosome and the cells became slender and polarized, adopting the so-called “comma-shape”. On the basis of these observations, the transformation process was classified into three steps: cell-nuclear migration, flagella formation and swarm maturation. The comigration of the cell nucleus and the centrosome was not inhibited by the anti-microtubule drug nocodazole (4 μM) but it was inhibited by the anti-microfilament drug cytochalasin A (4 μM), suggesting that the force of migration is generated by microfilaments. To investigate the role of the centrosome in this comigration in detail, we identified two aberrant strains, defective in swimming ability, from among various laboratory strains. The two strains, TM 4 and J, were found to have defects in cell-nuclear migration. Strain TM 4 had two types of irregular swarm cells: in one, only a part of the cell nucleus projected a thin filamentous structure; and in the other, no cell-nuclear migration occurred. Strain J had two centrosomes per cell and such swarm cells exhibited an attempt of cell-nuclear migration at two sites which corresponded to the position of the centrosome. The characteristics of these two strains indicate that the centrosome is essential for cell-nuclear migration. Our observations suggest that the cell-nuclear migration is mediated by actin-generated forces that act on the centrosome rather than on the cell nucleus itself.
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  • 6
    ISSN: 1615-6102
    Keywords: DNA-protein interaction ; Nicotiana tabacum ; Proplastid ; Proplastid-nuclei (nucleoids) ; Transcription
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have developed a novel assay system for analyzing the relationship between the structure and the transcriptional activity of the plastid-nuclei (plastid-nucleoids). The organization of morphologically intact proplastid-nuclei, isolated from tobacco cultured cells (line BY-2), was dispersed by treatment with NaCl at various concentrations and their transcriptional activities were examined by an assay of transcription in vitro and Southern hybridization. Disturbance of the structural organization of the proplastid-nuclei caused changes in both absolute and relative transcriptional activities of plastid genes, a result that suggests that the transcriptional activity of plastid genes may actually be regulated by structural changes in the plastid-nuclei.
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  • 7
    ISSN: 1615-6102
    Keywords: Mitochondrial nuclei ; Mitochondrial division ; Mitochondrial replicon cluster ; Physarum polycephalum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We investigated the manner of mitochondrial DNA (mtDNA) replication and distribution during the culture ofPhysarum polycephalum amoebae cells by microphotometry, anti-BrdU immunofluorescence microscopy, and quantitative hybridization analysis. In amoebae cells ofP. polycephalum, the number of mitochondria per cell and the shape of both mitochondria and mitochondrial nuclei (mt-nuclei) noticeably changed over the culture period. At the time of transfer, about 27 short ellipsoidal shaped mitochondria, which each contained a small amount of DNA, were observed in each cell. The number of mitochondria per cell decreased gradually, while the amount of mtDNA in an mt-nucleus and the length of mt-nuclei increased gradually. Midway through the middle logarithmic growth phase, the number of mitochondria per cell reached a minimum (about 10 mitochondria per cell), but most mtnuclei assumed an elongated shape and contained a large amount of mtDNA. During the late log- and stationary-growth phase, the number of mitochondria per cell increased gradually, while the amount of DNA in an mt-nucleus and mt-nuclei length decreased gradually. Upon completion of the stationary phase, the number and condition of mitochondria within cells returned to that first observed at the time of transfer. The total amount of mtDNA in a cell increased about 1.6-fold the first day, decreased immediately, then maintained a constant level ranging from 130 to 160 T. Except for the fact that mtDNA synthesis began earlier than synthesis of cell nuclei, the rate of increase in mtDNA paralleled that of cell-nuclear DNA throughout the culture. These results indicate that mtDNA is continuously replicated in pace with cell proliferation and the rate of mitochondrial division varies during culture; this mitochondrial division does not synchronize with either mtDNA replication or cell division. Furthermore, we observed the spatial distribution of DNA replication sites along mt-nuclei. Replication began at several sites scattered along an mt-nucleus, and the number of replication sites increased as the length of mt-nuclei increased. These results indicate that mtDNA replication progresses in adjacent replicons, which are collectively termed a mitochondrial replicon cluster.
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  • 8
    ISSN: 1615-6102
    Keywords: Brilliant sulfoflavin ; Cyanidioschyzon merolae ; Fenton reaction ; Fluorescence microscopy ; Hydrogen peroxide ; Microbody
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A novel procedure is described for fluorescence staining of microbodies, which can be applied quickly and easily. We developed this technique of microbody staining with the unicellular red algaCyanidioschyzon merolae. Cyanidioschyzon merolae only contains a single chloroplast, mitochondrion, and microbody per cell, and the mitotic cycle and the organelle division cycle are easily synchronized. Knowing that the concentration of H2O2 in the microbody is higher than it is in the cytosol and other cell components, we attempted to visualize the microbody by using fluorescence microscopy to detect H2O2. Brilliant sulfoflavin (BSF), used for detecting Fe2+ in analytical chemistry, fluoresces when it reacts with Fe2+ and H2C2. We were able to specifically stain microbodies with BSF, under acidic conditions (pH 3.0 or pH 2.5) with blue-light excitation. Using this procedure, we observed division of the microbody and the effect of aphidicolin on the microbody. We also discovered that microbody division is regulated by the cell nucleus and follows division of the cell nucleus.
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  • 9
    ISSN: 1615-6102
    Keywords: Chlamydomonas reinhardtii ; DAPI ; Basal bodies ; Absence of DNA ; Fluorimetry
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A search was made for DNA in both the basal bodies (BBs) in situ and BBs isolated from cells ofChlamydomonas reinhardtii by high-resolution epifluorescence microscopy after staining with 4′-6-diamidino-2-phenylindole (DAPI), by fluorimetry using a video-intensified microscope photon-counting system (VIMPICS) and by immunofluorescence microscopy after staining with a monoclonal tubulin-specific antibody. The flagella and intracellular microtubules radiate from the BBs. The BBs in young vegetative cells, gametes and young zygotes do not emit fluorescence after staining with DAPI but the spherical cell nucleus, the ovoid chloroplast nuclei and the tiny mitochondrial nuclei emit bright, blue-white fluorescence. Thus, it appears that BBs do not contain larger amounts of DNA than do the other organelles. To avoid the halation effects of fluorescence from the cell debris and cytoplasm and to measure carefully any extremely low levels of DNA that might be present in the organelles, a complex, composed of two flagella, a pair of BBs and the cell nucleus, was isolated from the gametes by treatment with autolysin and 0.1% Triton X-100. After staining with DAPI, the BBs of such complexes exhibit faint fluorescence while the cell nucleus emits strong fluorescence. The point and total intensities of the fluorescence emitted from each portion of the complex were measured with the VIMPICS. When the fluorescence intensity “T” of T 4 phage is taken as a standard, the fluorescence intensities of the flagella, the pair of BBs, the cell nucleus and the nucleus ofEscherichia coli are respectively 0.2 T, 0.40 T, 1452.2 T and 20.4 T. The slight fluorescence emitted from the BB seems to be due to the halation of the fluorescence emitted from the cell nucleus. The intensity of the fluorescence from the BBs is reduced to the intensity of the fluorescence of the flagella when the cell nucleus is removed from the complex. From these results, we conclude that the BBs do not contain DNA. Discrepancies related to the reported presence of DNA in the BBs are discussed.
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  • 10
    ISSN: 1615-6102
    Keywords: Organelle nuclei ; Maternal inheritance ; Pollen-specific nuclease ; Lilium longiflorum ; Pelargonium zonale
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4′,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca2+-dependent nuclease and a 23 kDa Zn2+ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.
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