Number Of Nucleoids Research Articles

The chloroplast nucleoids of the bundle sheath and mesophyll cells of Zea mays

Dimorphic chloroplasts of Zea mays L. cv. GH5004 from bundle sheath and mesophyll cells contained similar amounts of DNA, while bundle sheath chloroplasts contained twice the number of nucleoids compared to mesophyll chloroplasts. On average bundle sheath nucleoids were half the size of mesophyll nucleoids and contained half as much DNA. Electron microscope autoradiography of the chloroplasts showed that the nucleoid DNA is associated with the thylakoids and in the case of mesophyll chloroplasts preferentially with the grana. These observations suggest that the differences in nucleoid distribution may be due to differences in membrane morphology, with the small nucleoids of agranal bundle sheath chloroplasts being widely dispersed.

THE LACK OF CHLOROPLAST DNA IN ACETABULARIA MEDITERRANEA (ACETABULUM) (CHLOROPHYCEAE): A REINVESTIGATION

Three features of chloroplast DNA (cpDNA) in plastids isolated from Acetabularia mediterranea (acetabulum) were analyzed after staining the organelles with the fluorochrome 4′6-diamidino-2-phenyl indole (DAPI): (1) number of chloroplasts exhibiting DNA fluorescence, (2) number of nucleoids per plastid, and (3) nucleoid morphology. In vegetative Acetabularia cells only half of the total chloroplast population comprising several millions displayed the whitish-blue fluorescence of the DNA/DAPI complex. This percentage remained stable independent of whether cells were grown in supplemented natural sea water or enriched synthetic sea water. A single nucleoid, widely differing in size and morphology among the organelles, was characteristic of 76–81% of chloroplasts with DNA. Less than 20% contained two nucleoids, and in rare cases three or four nucleoids were present. The pattern of nucleoid numbers followed a Poisson distribution in one experiment, if calculated with the intrinsic mean of the observed data. In two other experiments, however, a significant difference existed between observed and expected values for a Poisson distribution according to the Chisquared test. After secondary enlargement of portions of the negatives, the nucleoids’substructure was disclosed and found to consist of brightly fluorescent spots interspersed by unstained regions The lack of cpDNA in Acetabularia cells appears to be brought about by (1) the polarized pattern of growth and translation confined to the apical region of the single cell and (2) the cpDNA arrangement in a single nucleoid acentrically located in the organelle. A scheme for the evolution of a chloroplast population having plastids without DNA is proposed. In theory the lack of cpDNA could arise in each plant, since chloroplasts never evolved a mitotic-like spindle to ensure the equal distribution of genetic material. The different nucleoid arrangement in most other plants, however, efficiently counteracts this ‘carelessness of nature’

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication. The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.

The effect of light on the number of chloroplast nucleoids in daughter cells of the algaScenedesmus quadricauda

Synchronous cultures of the green algaScenedesmus quadricauda (Turp.) Breb. grown at mean irradiances 25Wm−2, 75Wm−2, and 130Wm−2 PhAR were exposed to different illumination regimes (ratio of light to dark interval varied from 2:22 to 24:0 hours). The populations of daughter cells released under these conditions differed markedly in their progress in the cell cycle. The cells from these populations were stained with DAPI and the shape, localization and number of chloroplast nucleoids were examined. The nucleoids were of spherical shape, divided asynchronously having dumbbell shape during fission. In the chloroplast, nucleoids were located symmetrically about the transverse axis of the cells. The mean number of nucleoids varied from two in the least developed daughter cells to 16 in the daughter cells of the highest developmental stage. The progress of these cells and thus also the number of nucleoids were proportional to the portion of the light energy amount which these daughter cells shared from the total light energy amount obtained by their mother cells.

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm−2 to 130Wm−2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (2∶22 to 24∶0 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.

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 4'6-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.

The fate of chloroplast DNA during cell fusion, zygote maturation and zygote germination in Chlamydomonas reinhardi as revealed by DAPI staining

Chlamydomonas reinhardi, a haploid isogamous green alga, presents a classic case of uniparental inheritance of chloroplast genes. Since the molecular basis of this phenomenon is poorly understood, an examination of the cytology of the C. reinhardi plastid DNA was made in gametes, newly formed zygotes, maturing zygotes, and at zygote germination. The single plastid per cell of Chlamydomonas contains a small number of DNA aggregates (‘nucleoids’) which can be seen after staining with DNA-binding fluorochromes. In zygotes formed by pre-stained gametes, the fluorescing nucleoids disappear from the plastid of mating type minus (male) gamete plastids but not from the plastid of mating type plus (female) gamete plastids about 1 h after zygote formation. Subsequently, nucleoids aggregate slowly to a final average of two or three in the single plastid of the mature zygote. Quantitative microspectrofluorimetry indicates that gametes of both mating types have equal amounts of plastid DNA, and that zoospores arising from zygotes have 3.5 × as much as gametes. Assuming degradation of male plastid DNA, there must be a very major synthesis of plastid DNA between zygote formation and zoospore release when zygotes produce the typical 8–16 zoospores. That synthesis appears to occur at germination, where there is a massive increase in plastid DNA and nucleoid number beginning just prior to meiosis. The results support the theory that uniparental inheritance results from degradation of plastid DNA entering the zygote via the male gamete and suggest further studies, using mutants and altered conditions, which might explain how male plastid DNA sometimes survives.

Bacterial chromosome segregation: evidence for DNA gyrase involvement in decatenation.

Nucleoids isolated from a temperature-sensitive gyrB mutant of E. coli, incubated at restrictive temperatures, exhibit increased sedimentation rates and an abnormal doublet or dumbbell-shaped morphology. Shifting cells from restrictive to permissive temperature prior to nucleoid isolation leads to decreases in the percentage of doublet nucleoids and in nucleoid sedimentation rates. When nucleoids isolated from mutant cells exposed to restrictive temperature are incubated with purified gyrase, the percentage of doublet nucleoids decreases as the total number of nucleoids increases. These results, together with the demonstrated ability of gyrase to decatenate small circular DNA molecules in vitro, suggest that gyrase participates in bacterial chromosome segregation through its decatenating activity.

Cytological demonstration of chloroplast DNA behavior during gametogenesis and zygote formation in Chlamydomonas reinhardtii

We used the flourescent dye DAPI to visualize nucleoids of chloroplast DNA and follow their behavior through sexual reproduction by counting nucleoids in fixed cells at various stages. Nucleoid number varied greatly among cells at each stage. The mean number of nucleoids per cell was similar in mt (+) and mt (-) vegetative cells, and declined similarly during gametogenesis. Longer periods of nitrogen starvation reduced the mean nucleoid number further. Mean nucleoid number declined again in mating pairs, and continued to drop in zygotes up to the latest stage that can be examined (24-h zygotes). The oldest zygotes had means of about 2 to 3 nucleoids in different experiments, significantly fewer than in the mt (+) gametes (usually 4 to 5). The quantitative data on nucleoid number, mating efficiency, and germination efficiency allowed us to show that the decrease in nucleoid number is not limited to gametes that do not mate, or to zygotes that do not germinate. These data are consistant with earlier biochemical studies showing loss of chloroplast DNA during gametogenesis in both mating types, and with the degradation of paternal chloroplast DNA detected biochemically and (in non-quantitative studies) by DAPI staining. There may also be some fusion of nucleoids, although if it occurs it is not complete by 24 h of zygote maturation.

Cytological detection of the basis of uniparental inheritance of plastid DNA in Chlamydomonas moewusii

By using the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) to stain DNA one can follow the pattern of events affecting plastid DNA which occur in the formation and maturation of individual zygotes of the green flagellate Chlamydomonas moewusii. This species, like C. reinhardi, expresses uniparental inheritance of plastid DNA characters among zygote progeny, and is particularly favorable for cytological observation because the locale of the contribution of each gamete can still be recognized in mature zygotes. Gametes contribute equal numbers of DNA nucleoids, and amounts of plastid DNA (as measured by DAPI-DNA micro spectrofluorometry), to the zygote at fusion. Starting at nine hours, coincident with the further fusion of cell contents, plastid DNA disappears from the plastid contributed by one gamete. Further slow coalescence of nucleoids leads to a final nucleoid number per zygote approximately 1/3 of the sum of the 2 gametes.The DNA loss from one gamete plastid may require plastid contact to be initiated. Both light and nutrient availability affect the final number and distribution of plastid DNA nucleoids in the mature zygote. These observations are related to known genetic and biochemical data on uniparental inheritance of plastid characters.

Behavior of leucoplast nucleoids in the epidermal cell of onion (Allium cepa) bulb

The behavior of nucleoids during the leucoplast division cycle in the epidermis of onion (Allium cepa) bulbs was investigated using DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) staining. The leucoplast was morphologically amoeboid and continuously changed its shape. A dumbbell-shaped leucoplast divided into two spherical daughter ones by constriction in the middle region of the body. Leucoplasts contained 4–10 mostly spherical, oval, partly rodand dumbbell-shaped nucleoids which were dispersed within the bodies. The proportion of one DNA molecule of a T4 phage particle to the small leucoplast nucleoid in the grain density of negative film was 1 to 0.91. Comparison of the present result and another groups' biochemical results suggested that a small leucoplast nucleoid contains one DNA molecule. The dumbbell-shaped leucoplast probably before division contained about twice as many nucleoids as the spherical leucoplast after division, and each half of the dumbbell contained about half the number of nucleoids. Nucleoids increased in number with growth of the leucoplast. The behavior of nucleoids during the leucoplast division cycle in onion bulbs was basically similar to that during the chloroplast division cycle in higher plants and green algae, which was previously reported (Kuroiwa et al. 1981 b).

Conformation and segregation of nucleoids accompanying cell length extension after completion of a single round of DNA replication in germinated and outgrowing Bacillus subtilis spores.

When germinating spores of the temperature-sensitive DNA initiation mutant of Bacillus subtilis TsB134 are shifted to the restrictive temperature at a time such that just one or two rounds of replication are accomplished, the completed, nonreplicating nucleoids that form eventually adopt a doublet conformation. This conformation has now been observed after fixation by glutaraldehyde or osmium tetroxide, as well as by Formalin as found previously. The doublet was observed in media of different degrees of richness and under both light and electron microscopes. Electron micrographs of serial sections through the doublet were consistent with its formation by the gradual pulling apart of a single mass of DNA into two lobes. A systematic study was made of the effect of the time of shifting from the permissive to the restrictive temperature and of the restrictive temperature used on the number of nucleoids segregating within the outgrowing rod. It was established that the doublet nucleoid behaved as a single unit in replication control and segregation in both rich and poor media. Measurement of the relative position of the two segregating nucleoids within the outgrowing rod after completion of just one round of replication yielded quantitative information on the segregation and cell length extension processes. Segregation was accompanied by cell length extension at approximately equal rates on both sides of each nucleoid. Furthermore, the data were consistent with an exponential increase in such an extension with time over the early and major portion of the period studied, but it was not possible to rule out other models of length extension.

Participation of the intracellular enzymes in the control of mutational processes

The influence of UV-specific endonuclease and medium composition on the frequency and spectrum of genic mutations in Escherichia coli KI2 uvr (+) (with normal repair enzymes) and urv A6 (defective in UV-specific endonuclease) was studied. Mutations at the locus glu (gene controlling assimilation of glucose) were induced by ultra-violet irradiation and hydroxylamine treatment. To identify mutant colonies, triphenyl tetrazolium chloride (TTC) was added to the medium since it coloured the mutant colonies bright crimson and readily permitted distinction between pure mutant clones (complete mutations) and mixed clones (mosaic or sector mutations).A maximum mutation frequency after UV-irradiation was observed in E. coli uvr (+) cells but not in the E. coli uvr A6 strain. The curve of mutagenesis with a maximum was found in both studied strains after treatment by hydroxylamine which did not cause DNA damage recognized by UV-specific endonuclease.The highest frequency of mutations (at the point of maximum) in the series of experiments with enriched growth medium was almost 10 times higher than in the series of the experiments with poor medium.It was established that in bacteria with normal repair enzymes the frequency of complete mutations was higher than the frequency of mosaic mutations. It was also observed that the rate of UV-mutagenesis was higher in the case of E. coli uvr (+).The study of the distribution of mosaic mutant sectors in experiments with bacteria suspended in either a nutrient broth or a buffer during UV-irradiation revealed that the size of mutant sectors was rather variable and that the differences in the number of nucleoids per cell did not always determine the distribution of mutant sector sizes.

Microscopic studies of Streptomyces hygroscopicus growth kinetics

Growth kinetics, branch formation, and cytological properties of mycelial growth of Streptomyces hygroscopicus on solid media were investigated by phase-contrast microscopy using a microculture method. Measurements were made on taken photographs of the growing hyphae from the beginning of spore germination up to maximal 18 hours. The specific growth rate of the germ tube was much higher than the specific growth rate of the mycelium. The doubling time of the total length of the mycelial hyphae and the doubling time of branch formation was quite the same for the period investigated. After a short time of outgrowth each individual hypha grows at a constant rate, i.e. the length increases linearly, but the growth kinetics of the whole mycelium becomes exponentially by branching. It seems, that nucleoids only divide in that part of a hypha between the tip and the nearest branch. A cell unit (1.4-1.9 mum on different media) could be calculated by the length of a hypha and the number of nucleoids. A hypha is growing with the cell unit at the tip or with the polar cap only. No interkalary growth in length could be found. Branches were formed only up to about 100 mum from the tip on complex medium and up to about 50 mum from the tip on mineral salt medium. A simple method of mycelial growth has been developed. Some properties showing the connection between Streptomycetes and prokaryotic organisms on one hand and hyphal growing fungi on the other hand are discussed.

Mikroskopische Untersuchungen zur Wachstumskinetik von Streptomyces hygroscopicus

AbstractGrowth kinetics, branch formation, and cytological properties of mycelial growth of Streptomyces hygroscopicus on solid media were investigated by phase‐contrast microscopy using a microculture method. Measurements were made on taken photographs of the growing hyphae from the beginning of spore germination up to maximal 18 hours. The specific growth rate of the germ tube was much higher than the specific growth rate of the mycelium. The doubling time of the total length of the mycelial hyphae and the doubling time of branch formation was quite the same for the period investigated. After a short time of outgrowth each individual hypha grows at a constant rate, i.e. the length increases linearly, but the growth kinetics of the whole mycelium becomes exponentially by branching. It seems, that nucleoids only divide in that part of a hypha between the tip and the nearest branch. A cell unit (1.4–1.9 μm on different media) could be calculated by the length of a hypha and the number of nucleoids. A hypha is growing with the cell unit at the tip or with the polar cap only. No interkalary growth in length could be found. Branches were formed only up to about 100 μm from the tip on complex medium and up to about 50 μm from the tip on mineral salt medium. A simple model of mycelial growth has been developed. Some properties showing the connection between Streptomycetes and prokaryotic organisms on one hand and hyphal growing fungi on the other hand are discussed.

A Change in the Number of Nucleoids inProteus vulgaris during a Penicillin-induced L-cycle

The author studied changes in the number of nucleoids inProteus vulgaris in the course of a penicillin-induced incomplete L-cycle. It was found that the number of nucleoids increased significantly during the development of large bodies from rods, and more so on solid than in liquid media. The size of the nucleoids did not decrease, but remained the same during the whole L-cycle (0.2–0.3 μ). Most nucleoids are spherical, though some are rod-shaped. In older large bodies they give the impression of a reticular arrangement.

The bacterial genus Lineola.

SUMMARY: Lineola, a new bacterial genus, is characterized by large and very long cells of an unusual type and by the formation of motile trichomes of considerable length which may be branched. Two species were isolated in pure culture by the use of capillary pipettes. Even when their nutritional requirements were known it was difficult to purify them by plating. The growths of the two species on agar are different because the trichomes of L. articulata are much softer than those of L. longa. The cells of both species contain a large number of nucleoids and divide by constriction. In L. longa the daughter cells remain firmly connected, whereas in L. articulata the links become pliable, the whole chain-like trichome bending when swimming. Both species are Gram-negative and have numerous flagella. No spores were found. Media with peptone, meat extract, yeast extract, and the like must be supplemented with acetate and with agar or certain other substances, to support multiplication.