Structure,composition, and distribution of plastid nucleoids in Narcissus pseudonarcissus

The size, frequency and distribution of the nucleoids of chloroplasts (cl-nucleoids) and chromoplasts (cr-nucleoids) of the daffodil have been investigated in situ using the DNA-specific fluorochrome 46-diamidino-2-phenylindole. Chromoplasts contain fewer nucleoids (approx. 4) than chloroplasts (> 10), and larger chromoplasts (cultivated form, approx. 4) contain more than smaller ones (wild type, approx. 2). During chromoplast development the nucleoid number decreases in parallel with the chlorophyll content. Each nucleoid contains 2–3 plastome copies on average. In chloroplasts the nucleoids are evenly distributed, whereas they are peripherally located in chromoplasts. The fine structure of isolated cl-and cr-nucleoids, purified either by Sepharose 4B-CL columns or by metrizamide gradients, was investigated electron microscopically. The cl-nucleoids consist of a central protein-rich core with naked DNA-loops protruding from it. In cr-nucleoids, on the other hand, the total DNA is tightly packed within the proteinaceous core. The protein-containing core region of the nucleoids is made up of knotty and fibrillar sub-structures with diameters of 18 and 37 nm, respectively. After proteinase treatment, or incressing ion concentration, most of the proteins are removed and the DNA is exposed even in the case of cr-nucleoids, the stability of which proved to be greater than that of cl-nucleoids. The chemical composition of isolated plastid nucleoids has been determined qualitatively and quantitatively. Chromoplast-nucleoids contain, relative to the same DNA quantity, about six times as much protein as cl-nucleoids. Accordingly the buoyant density of cr-nucleoids in metrizamide gradients is higher than that of cl-nucleoids. In addition to DNA and protein, RNA could be found in the nucleoid fraction. No pigments were present. The cr-and cl-nucleoids have many identical proteins. There are, however, also characteristic differences in their protein pattern which are possibly related to the different expression of the genomes of chloroplasts and chromoplasts. Nucleoids of both plastid types contain some proteins which also occur in isolated envelope membranes (probably partly in the outer membrane) and thus possibly take part in binding the DNA to membranes.Abbreviations cl- chloroplast - cr- chromoplast - DAPI 46-diamidino-2-phenylindole - DNase deoxyribonuclease - kDa kilodaltons - MG purified by metrizamide gradients - SC purified by Sepharose CL-4B column gel filtration - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Urate-mediated regulation of urate oxidase inChlamydomonas reinhardtii

Summary Expression of uncase (urate oxidase) fromChlamydomonas reinhardtii has been investigated by using specific polyclonal antibodies. By Western blot analyses performed under nondenaturing conditions, a 124 kDa protein band corresponding to active uricase was detected in protein extracts from cells cultured with urate or nitrogen-starved cells. This protein band was absent in cells cultured with ammonium. Besides the 124 kDa band, the antibodies also reacted with a massive protein band, with an apparent molecular mass of 500 kDa, that was detected in all nutritional conditions assayed. In vitro, inactive uricase from cells grown with ammonium was activated by incubation in presence of urate. The appearance of uricase activity in vitro coincided with a decrease of the 500 kDa protein and an increase of the 124 kDa band corresponding to the active enzyme. We suggest that a posttranslational regulation of uricase synthesis takes place inC. reinhardtii, and that urate may be responsible for the assembly or maturation of inactive precursors to form the active uricase.     

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Summary Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.Abbreviations DAPI 4,6-diamidino-2-phenylindol - DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Disappearance of plastid and mitochondrial nucleoids during the formation of generative cells of higher plants revealed by fluorescence microscopy

Summary The fate of plastid and mitochondrial nucleoids (pt and mt nucleoids) ofTriticum aestivum was followed during the reproductive organ formation using fluorescence microscopy after staining with 4'6-diamidino-2-phenylindole (DAPI). This investigation showed a drastic morphological change of pt nucleoids during the differentiation of reproductive organs from the shoot apex. Dot-shaped pt nucleoids grew into ring-shaped ones, which divided into small pieces in the monocellular pollen grain, as observed in this plant's earlier stage of leaf development. During the development of mature pollen grain from monocellular pollen grain, pt and/or mt nucleoids disappeared through the division of the male generative cell ofT. aestivum. Cytologically, this observation is direct evidence of the maternal inheritance of higher plants. Thus far, cytological evidence of this phenomenon has been found mostly by morphological criteria using electron microscopy, which admits some ambiguity. In the plants exemplified byLilium longiflorum, pt and/or mt nucleoids disappeared after the first pollen grain mitosis, which precededT. aestivum. In the plants exemplified byTrifolium repens, pt and/or mt nucleoids existed in the generative cells of the mature pollen grain.The significance of these observations was discussed in relation to the interaction between nuclear and organelle genomes during plant development.Abbreviations DAPI 4'6 diamidino-2-phenylindole - Mt DNA Mitochondrial DNA - Mt nucleoid Mitochondrial nucleoid - Pt DNA Plastid DNA - Pt nucleoid Plastid nucleoid On leave from Department of Biology, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

Chloroplast nucleoids in large number and large DNA amount with regard to maternal inheritance inChlamydomonas reinhardtii

Summary We studied the maternal chloroplast inheritance ofChlamydomonas reinhardtii by epifluorescence microscopy after staining with DNA specific fluorochrome DAPI and by genetic methods, using wild type cells and cells containing previously isolated mutation of cond-1 and cond-2. Wild type cells contained about 7 chloroplast (cp) nucleoids, while mutants, cond-1(+) and cond-2(+), contained about 14 and 23 cp nucleoids, respectively, after one week culture on agar plates. The total cpDNA contents were almost proportional to the numbers of cp nucleoids. When cells containing cond-1 or cond-2 mutation were used as a parental source to cross with wild type cells of the other parent, preferential digestion of cp nucleoids from male parent (mt^–) origin occurred in the zygotes, although the frequencies of the digestion were slightly lower than that in the zygotes from the cross between wild type cells. Western blot analysis of the protein ofzyslB gene, which has been found related to preferential digestion of mt^– origin cp-nucleoids DNA, showed that a high amount of this protein was detected with the initiation of preferential digestion of mt^– cp nucleoids and disappeared with the completion of the digestion. Cp genetic markers for antibiotic resistance were maternally inherited in all crosses. These results showed that although the preferential digestion of cp nucleoids consisting of large number and large cpDNA amount requires a slightly longer period to complete, this high ploidy of the cp nucleoids does not disturb maternal inheritance.     

Xanthine accumulation and vacuolization inChlamydomonas reinhardtii cells

Summary Utilization of xanthine as the sole nitrogen source for growth byChlamydomonas reinhardtii cells involved the formation of a transient, intracellular pool of xanthine. Up to 20% of the total xanthine supplied to the medium was not assimilated after uptake but stored in the cells at concentrations that exceeded xanthine solubility in water. At the subcellular level, a massive accumulation of starch grains in the chloroplast and the appearance of many vacuoles in the cytoplasm distinguished xanthine-grown from ammonium-grown cells. Starch accumulation, but not development of vacuoles, was also observed in N-starved cells. Uptake experiments with radio-labelled xanthine showed that this accumulates only in the cytoplasm, most probably inside vacuoles. The electron-dense material observed in vacuoles of xanthine-grown cells suggests that the intracellular xanthine is in part solid xanthine.     

Sexual agglutinin of mating-type minus gametes inChlamydomonas reinhardtii

The sexual agglutinin from the mating-type minus gametes ofChlamydomonas reinhardtii was purified by gel filtration and hydroxyapatite chromotography. The minus agglutinin was identified as a single glycopolypeptide termed Agg(-) of very high molecular weight by SDS-poly-acrylamide gel electrophoresis. It was also observed as a glycoprotein with agglutinin activity on non-SDS polyacrylamide gels. The native agglutinin appeared to be composed of a complex of Agg(-) subunits. It consisted of about 60% protein and 40% carbohydrate and the activity was diminished by a mild periodate oxidation. When the plus agglutinin was also purified and compared with the minus agglutinin, it was found that both agglutinins migrate in the same position by SDS-polyacrylamide gel electrophoresis, whereas their behaviors on gel filtration and hydroxyapatite chromatography are different.Abbreviations mt ^+/– mating-tape plus or minus - SDS sodium dodecyl sulfate - V_e elution volume - V_o void volume - kDa kilodalton     

Preferential digestion of chloroplast nuclei (nucleoids) during senescence of the coleoptile ofOryza sativa

Summary The coleoptile ofOryza sativa develops, grows and ages within 4 days that follow imbibition. It is, thus, a very useful system for experimental analysis of the life cycle of organelles, for example, the development, growth and aging of plastids in higher plants. We examined the behavior and levels of DNA and chlorophyll in the plastid by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified-photon counting system (VIMPCS). The whitish yellow coleoptile appeared soon after imbibition and, between the first 24 and 60 h that followed imbibition, it grew markedly in a longitudinal direction, with concomitant elongation of the cells, and an increase in the volume of plastids and in the amount of DNA in the plastids. The chlorophyll content per plastid began to increase when the coleoptile turned green, 48 h after imbibition, and reached a plateau value when the coleoptile was 3.5 mm in length, 72 h after imbibition. More than 12 h later, the chlorophyll disappeared just before the breakdown of chloroplasts was initiated. Proplastids in young coleoptiles, contained a plastid nucleus which was located in the central area of the plastids and each nucleus consisted of approximately 6 copies of plastid DNA (ptDNA). The number of copies of ptDNA per plastid increased gradually, with a concomitant increase in the volume of the plastids after imbibition, and reached approximately 130 times the value in the young proplastids, 60 h after imbibition, when the plastid developed into a chloroplast. However, each plastid nucleus did not scatter throughout the entire interior region of each chloroplast. The disappearance of each plastid nucleus occurred more than 12 h before the degeneration of the chloroplasts. The number of plastids per cell increased from 10 to 15 in young coleoptiles within 12 h after imbibition. Yet the number remained constant throughout subsequent growth and aging of the coleoptile. Thus the preferential reduction in the amount of chloroplast DNA was not due to the division of the plastid but could, perhaps, be associated directly with the aging of the cells of the coleoptile which precedes senescence of the coleoptiles.     

Quantitative study of organelle nucleoids during the second pollen grain mitosis inOenothera biennis

Summary The behavior of organelle nucleoids in the generative cell was examined at the second (pollen grain) mitosis by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) inOenothera biennis. TheO. biennis generative cell contained a large number of organelle nucleoids distributed randomly in the cytoplasm before mitosis. The epifluorescence images of the nucleoids could be classified distinctly into two groups which corresponded to plastid nucleoids (pt-nucleoids) and mitochondrial nucleoids (mt-nucleoids). Discrimination between pt- and mt-nucleoids was carried out with the aid of DNA immunogold electron microscopy. At metaphase, both pt- and mt-nucleoids migrated to the pole regions of the generative cell. After mitosis, organelle nucleoids in both of the sperm cells scattered in the cytoplasm again. A quantitative examination of pt-nucleoids on 202 pairs of sperm cells showed that the leading sperm cell (Svn) contained 0–39 pt-nucleoids (19.0 ± 7.4) and the trailing sperm cell (Sua) contained 0–40 pt-nucleoids (15.4 ± 6.5). For mt-nucleoids, examination of 28 pairs of sperm cells showed that Svn contained 5–32 mt-nucleoids (14.5 ± 6.8) and Sua contained 6–30 mt-nucleoids (13.4 ±7.5). These results showed that (1) the number of organelle nucleoids per sperm cell varied considerably in the cells studied; (2) quantitative difference in pt- and mt-nucleoids between Svn and Sua could occur in some gametophytes studied; but (3) it was unlikely that there was any pre-differentiational cytoplasm localization and essential sperm heteromorphy with respect to organelle nucleoid content in the gametophyte population.     

Changes in distribution of nucleoids in developing and dividing chloroplasts and etioplasts ofArena sativa

Summary Nucleoid distribution in chloroplasts and etioplasts at the different developmental stages was examined with the first leaves ofAvena sativa by using a DNA-specific fluorescent probe, 46-diamidino-2-phenylindole (DAPI). In light-grown first leaves, three types of plastid nucleoid distribution were recognized. 1. Peripheral distribution in undeveloped chloroplasts which contain only a few thylakoids in the middle region of the leaf sheath. 2. Ring-like arrangement along the rim of developing and dividing young chloroplasts, of which grana were composed of four to eight layers of thylakoids, at the base of the leaf blade. The plane of the nucleoids' ring is in parallel with the face of the thylakoids. 3. Scattered distribution of 10 to 20 discrete spherular nucleoids in the stroma of fully developed chloroplasts, of which grana were composed of up to 20 thylakoids, in the regions of the middle and the tip of the leaf blade. In dark-grown first leaves two types were recognized. 1. Peripheral distribution in developing and dividing young etioplasts in the leaf sheath and the base of the leaf blade. 2. Scattered distribution of 10 or more discrete spherular nucleoids in fully developed etioplasts, containing extended prothylakoids, in the regions of the middle and the tip of the leaf blade. Ring-like arrangement of nucleoids was not observed in any etioplasts. The results indicates that spatial arrangement of plastid nucleoids dynamically changes in close relationship with the development of the inner membrane systems of plastids.     

Biochemical,genetic and molecular characterization of new respiratory-deficient mutants inChlamydomonas reinhardtii

Eight respiratory-deficient mutants ofChlamydomonas reinhardtii have been isolated after mutagenic treatment with acriflavine or ethidium bromide. They are characterized by their inability to grow or their very reduced growth under heterotrophic conditions. One mutation (Class III) is of nuclear origin whereas the seven remaining mutants (Classes I and II) display a predominantly paternalmt ^- inheritance, typical of mutations residing in the mitochondrial DNA. Biochemical analysis has shown that all mutants are deficient in the cyanide-sensitive cytochrome pathway of the respiration whereas the alternative pathway is still functional. Measurements of complexes II + III (antimycin-sensitive succinate-cytochromec oxido-reductase) and complex IV (cytochromec oxidase) activities allowed to conclude that six mutations have to be localized in the mitochondrial apocytochromeb (COB) gene, one in the mitochondrial cytochrome oxidase subunit I (COI) gene and one in a nuclear gene encoding a component of the cytochrome oxidase complex. By using specific probes, we have moreover demonstrated that five mutants (Class II mutants) contain mitochondrial DNA molecules deleted in the terminal end containing the COB gene and the telomeric region; they also possess dimeric molecules resulting from end-to-end junctions of deleted monomers. The two other mitochondrial mutants (Class I) have no detectable gross alteration. Class I and Class II mutants can also be distinguished by the pattern of transmission of the mutation in crosses.Anin vivo staining test has been developed to identify rapidly the mutants impaired in cyanide-sensitive respiration.     

Regulation of carbonic-anhydrase activity,inorganic-carbon uptake and photosynthetic biomass yield inChlamydomonas reinhardtii

The regulation of carbonic anhydrase by environmental conditions was determined forChlamydomonas reinhardtii. The depression of carbonic anhydrase in air-grown cells was pH-dependent. Growth of cells on air at acid pH, corresponding to 10 m CO₂ in solution, resulted in complete repression of carbonic-anhydrase activity. At pH 6.9, increasing the CO₂ concentration to 0.15% (v/v) in the gas phase, corresponding to 11 M in solution, was sufficient to completely repress carbonic-anhydrase activity. Photosynthesis and intracellular inorganic carbon were measured in air-grown and high-CO₂-grown cells using a silicone-oil centrifugation technique. With carbonic anhydrase repressed cells limited inorganic-carbon accumulation resulted from non-specific binding of CO₂. With air-grown cells, inorganic-carbon uptake at acid pH, i.e. 5.5, was linear up to 0.5 mM external inorganic-carbon concentration whereas at alkaline pH, i.e. 7.5, the accumulation ratio decreased with increase in external inorganic-carbon concentration. It is suggested that in air-grown cells at acid pH, CO₂ is the inorganic carbon species that crosses the plasmalemma. The conversion of CO₂ to HCO ₃ ^- by carbonic anhydrase in the cytosol results in inorganic-carbon accumulation and maintains the diffusion gradient for carbon dioxide across the cell boundary. However, this mechanism will not account for energy-dependent accumulation of inorganic carbon when there is little difference in pH between the exterior and cytosol.     

Distribution and dynamics of mitochondrial nucleoids in animal cells in culture

Mitochondrial (mt) nucleoids were visualized in living cells in culture by staining with the fluorochrome picoGreen. The cell types included a line derived from Xenopus heart endothelial cells (XTH-2), 3T3 cells, SV40-transformed 3T3 cells and primary cultures of Xenopus tadpole epidermis cells. In the permanent cell lines 6-60% of the mitochondria were found to be devoid of DNA. The peaks of the frequency distribution of mtDNA content, as revealed by microfluorometry, were not very distinct, indicating the presence of a high amount of aneuploid mt nucleoids. The maximum size of nucleoids (as derived from fluorescence intensity) was 10-12 times that of the minimum peak value in proliferating cell cultures. A linear ratio was found between the volume of the nucleoids and their DNA content, which is interpreted as a uniform package density. In terminally differentiating tadpole epidermis cells mitochondria form large bodies containing giant nucleoids, while in mitotic cells the mt nucleoids are small and of uniform size. Fusion and fission of the nucleoids were observed to occur either for no visible reason or in connection with fusion and fission events of the mitochondria.Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Evolution of fragmented mitochondrial ribosomal RNA genes inChlamydomonas

The fragmented mitochondrial ribosomal RNAs (rRNAs) of the green algaeChlamydomonas eugametos andChlamydomonas reinhardtii are discontinuously encoded in subgenic modules that are scrambled in order and interspersed with protein coding and tRNA genes. The mitochondrial rRNA genes of these two algae differ, however, in both the distribution and organization of rRNA coding information within their respective genomes. The objectives of this study were (1) to examine the phylogenetic relationships between the mitochondrial rRNA gene sequences ofC. eugametos andC. reinhardtii and those of the conventional mitochondrial rRNA genes of the green alga,Prototheca wickerhamii, and land plants and (2) to attempt to deduce the evolutionary pathways that gave rise to the unusual mitochondrial rRNA gene structures in the genusChlamydomonas. Although phylogenetic analysis revealed an affiliation between the mitochondrial rRNA gene sequences of the twoChlamydomonas taxa to the exclusion of all other mitochondrial rRNA gene sequences tested, no specific affiliation was noted between theChlamydomonas sequences andP. wickerhamii or land plants. Calculations of the minimal number of transpositions required to convert hypothetical ancestral rRNA gene organizations to the arrangements observed forC. eugametos andC. reinhardtii mitochondrial rRNA genes, as well as a limited survey of the size of mitochondrial rRNAs in other members of the genus, lead us to propose that the last common ancestor ofChlamydomonas algae contained fragmented mitochondrial rRNA genes that were nearly co-linear with conventional rRNA genes.     

Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2

Unicellular algae grow well under limiting CO₂ conditions, aided by a carbon concentrating mechanism (CCM). In C. reinhardtii, this mechanism is inducible and is present only in cells grown under low CO₂ conditions. We constructed a cDNA library from cells adapting to low CO₂, and screened the library for cDNAs specific to low CO₂-adapting cells. Six classes of low CO₂-inducible clones were identified. One class of clone, reported here, represents a novel gene associated with adaptation of cells to air. A second class of clones corresponds to the air-inducible periplasmic carbonic anhydrase I (CAH1). These clones represent genes that respond to the level of CO₂ in the environment.     

Isolation of giant mitochondrial nucleoids from the yeastSaccharomyces cerevisiae

Summary The yeast cellsSaccharomyces cerevisiae grown up to stationary phase under either anaerobic conditions, or aerobic conditions in the presence of a respiratory inhibitor, antimycin A, had distinctive giant mitochondrial nucleoids (mt-nucleoids) (apparent diameter 0.6–0.9 m) in contrast with the small mt-nucleoids (apparent diameter 0.2–0.4 m) in respiratory-sufficient cells grown aerobically, as revealed by DAPI-fluorescence microscopy. The cytoplasmic respiratory-deficient cells (rho^– cells), which were induced by treatment of wild-type cells with ethidium bromide, showed both giant and small mt-nucleoids of irregular size. In order to examine the structural and functional differences between giant and small mt-nucleoids, the former were successfully isolated from spheroplasts of three different cells by differential centrifugation and centrifugation on a discontinuous sucrose gradient. The isolated giant mt-nucleoids were intact in the morphology and were free of significant contamination by nuclear chromatin. The number of protein components involved in each of three different giant mt-nucleoids was similar to the number in small mt-nucleoids from aerobically grown cells, though a few noticeable differences were also recognized. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa, 50 kDa, 38 kDa, 26 kDa, and 20 kDa were the main components of small mt-nucleoids from aerobically grown cells as detected by chromatography on native DNA-cellulose. In contrast, the 67 kDa and 52 kDa proteins were hardly detected in corresponding fractions of giant mt-nucleoids from anaerobically grown cells and from rho^– cells grown aerobically. On the other hand, mt-nucleoids from aerobically grown cells in the presence of antimycin A seemed to lack the 67 kDa protein but to have a small amount of the 52 kDa protein. This is the first demonstration of the variance of protein species involved in yeast mt-nucleoids according to the respiratory activity of mitochondria.     

Quantitative fluorescence microscopy on dynamic changes of plastid nucleoids during wheat development

Summary Dynamic change of plastid nucleoids (pt nucleoids) was followed by fluorescence microscopy after staining with 46-diamidino-2-phenyl indole (DAPI). The fluorescence image was quantified with a supersensitive photonic microscope system based on photon counting and image analysis. The results showed that small pt nucleoids located in the center of proplastids in the dry seed increased in size after imbibition and formed highly organized ring structures in the dark, which divided into ca. 10 pieces within 3 days. Corresponding to this morphological change, DNA content of a plastid multiplied 7.5 fold. Total increase in DNA content of pt nucleoids per cell was 34 times as that of dry seed, as plastid multiplied 4.6 times in the average during this period. Upon light illumination small pt nucleoids having basic genome size were separated from divided pt nucleoids, suggesting a relationship with the formation of thylakoid system. The significance of the procedure established in this study is discussed in analysing the dynamic changes of intracellular small genomes.On leave from Department of Biology, Faculty of Science, Nagoya University, Furocho, Chikusaku, Nagoya 464, Japan.     

The cell wall of Chlamydomonas reinhardtii gametes: Composition,structure and autolysin-mediated shedding and dissolution

The cell walls of Chlamydomonas gametes are multilayered structures supported on frameworks of polypeptides extending from the plasma membrane. The wall-polypeptide catalogue reported by Monk et al. (1983, Planta 158, 517–533) and extended by U.W. Goodenough et al. (1986, J. Cell Biol. 103, 405–417) was re-evaluated by comparative analysis of mechanically isolated cell walls purified from several strains. The extracellular locus of wall polypeptides was verified by in vivo iodogen-catalysed iodination and by autolysin-mediated elimination of the bulk of these polypeptides from the cell surface. Three (w15, w16, w17) and possibly four (w14) polypeptides were located to the most exterior aspect of the wall because of their susceptibility to Enzymobeadcatalysed iodination and their retention by a cell-wall-less mutant. The composition of shed walls stabilised with ethylenediaminetetraacetic acid during natural mating and kinetic analysis of the dissolution of walls purified from a bald-2 mutant demonstrated the rapid and specific destruction of polypeptide w3. Differential solubilisation of wall polypeptides occurred after loss of w3. Wall dissolution, characterised by the generation of fishbone structures from the W2 layer, gave as many as four additional polypeptides. Charged detergents and sodium perchlorate extracted a comparable range of polypeptides at room temperature from mechanically isolated walls, i.e. components of the W4–W6 layers, hot sodium dodecyl sulphate solubilised framework polypeptides, while reducing agent was required to solubilise the W2 layer. A model of wall structure is presented.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol