DNA-specific Fluorochrome 4 ' ,6-diamidino-2-phenylindole Research Articles

Studies on Cell Number and Nuclei in Spores and on Ploidy Level in Ascochyta rabiei Isolates

Isolates of the phytopathogenic ascomycete Ascochyta rabiei (Pass.) Labr. were stained with the DNA‐specific fluorochrome 4′,6‐diamidino‐2‐phenylindole (DAPI) and compared for differences in number of nuclei per pycnidiospore and the ploidy level. Microscopic analyses revealed that within the examined isolates five different combinations of cell number and number of nuclei in spores exist. A one‐celled spore may contain one, two and four nuclei, respectively, and in the case of two‐celled spores there exist types with one and two nuclei in one cell. Microfluorometric analyses of wild types and benomyl‐treated isolates revealed differences in ploidy level among the wild types.

Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.

Effect of fluorochromes on bacterial surface properties and interaction with granular media.

Simple, efficient, and safe tagging methods are desired in short-term microbial transport studies such as in the study of filtration systems for water and wastewater treatment. Suitability of selected fluorochromes as bacterial tagging agents in transport studies was evaluated on the basis of stability of stained cells and the effect of staining on bacterial surface characteristics and interaction with granular media. Surface properties were characterized by zeta potential and microbial adhesion to hydrocarbons. The effect of staining on interactions between bacteria and porous media was evaluated in terms of removal of bacteria in batch adsorption tests using sand coated with aluminum hydroxide to enhance adsorption. The DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) had generally negligible effects on bacterial surface properties and interaction with sand, as indicated in batch adsorption tests using pure cultures (Escherichia coli or Acinetobacter sp.) and wastewater bacteria. Cells stained with DAPI were stable for 48 h at 4 or 20 degrees C. Other nucleic acid fluorochromes tested had different but significant effects on bacterial cells and produced less stable fluorescence. Since transport through porous media is modulated by surface properties, it may be concluded based on these results that the choice of fluorochromes is critical in microbial transport studies. DAPI appeared to be a promising tagging agent. Time dependence of fluorescence of stained cells may limit the use of fluorochrome-tagged cells in long-term transport studies.

DNA synthesis and the cell cycle in the noxious red-tide dinoflagellate Gymnodinium nagasakiense

Gymnodinium nagasakiense is a noxious red tide dinoflagellate often associated with damage to fisheries in Japan. DNA synthesis and the cell cycle in this organism were investigated from 1989 to 1990 by determining relative DNA contents of individual cells using an epifluorescence microscopy-based microfluorometry system. The nuclei were stained with the DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI). Because photosynthetic pigment interferes with the fluorescence from the DAPI-DNA complex, the pigment was eliminated by methanol treatment as a first step in quantitative microfluorometry. Nuclear DNA contents, cell size distribution, cell density, and frequency of paired cells were determined every 2h for 24h using cells grown on a 12h light:dark cycle. DNA synthesis and cell division were tightly phased to a particular period of the light:dark cycle. DNA synthesis (S phase) occurred from 10:00 to 22:00 hrs and was followed by cytokinesis. The presence of such a distinct S phase strongly suggests thatG. nagasakiense has a typical eukaryotic cell cycle. This type of cell cycle makes it possible to estimate speciesspecific in situ growth rate based on the diel pattern of DNA synthesis.

Simultaneous measurement of DAPI-sulforhodamine 101 stained nuclear DNA and protein in higher plants by flow cytometry.

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.

DNA is present in the pyrenoid core of the siphonous green algae of the genusCaulerpa and yellow-green algae of the genusPseudodichotomosiphon

The pyrenoids of a number of siphonous green and yellow-green algae have been examined for the presence of chloroplast DNA (chloroplast nucleoids). We have found a conspicuous localization of chloroplast DNA in the pyrenoid core ofCaulerpa lentillifera andC. fergusonii (siphonous green algae), andPseudodichotomosiphon constrictus (a siphonous yellow-green alga) using fluorescence microscopy after staining with the DNA specific fluorochrome 4′,6-diamidino-2-phenylindole (DAPI).

Production of interferon-β upon induction with polyinosinic acid:polycytidylic acid during the cell cycle of human fibroblasts

The production of interferon-β was examined at various stages of the cell cycle in synchronized and unsynchronized cell populations induced by poly(I):poly(C). Human fibroblasts were synchronized with mitotic detachment and, at different stages of the cell cycle, poly(I):poly(C) was added for induction of interferon-β. One hour after induction, cell-free medium was collected and assayed for secreted interferon-β. The cells were then fixed and stained with a DNA-specific fluorochrome, 4′,6-diamidino-2-phenylindole (DAPI), for cell cycle analysis by microfluorometry. The data indicated that interferon-β was produced in every stage examined of the cell cycle. In addition, the level of intracellular interferon-β was quantitatively measured in single cells of an unsynchronized cell population using a specific antibody. In the same individual cell, DAPI fluorescence intensity was measured for determination of the cell cycle position. The results show that interferon-β protein can be detected throughout the cell cycle.

Variations in Morphology and DNA Distribution Among Chloroplasts in Mature Germling Cell of Bryopsis plumosa (Hudson) C. Ag. (Chlorophyceae)

Fluorescence microscopy with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) revealed the presence of many intensely fluorescing areas around the giant primary nucleus in the mature germling cell of the green alga Bryopsis plumosa. Each of the fluorescent areas was irregular in shape and larger and brighter than the nucleoids scattered uniformly within the chloroplasts which could ordinarily be seen in the germling cell. Near the primary nucleus there were 40 or more small lenticular chloroplasts. They were morphologically different from the usual fusiform chloroplasts and were generally poor in starch and often lacked pyrenoids. The intensely fluorescing areas were always associated with these small perinuclear chloroplasts, implying that they were nucleoids of these chloroplasts. Our results indicate that, among the chloroplasts in the germling cell of B. plumosa, those located near the giant primary nucleus differ from others in morphology and also in DNA distribution.

Presence of DNA in the pyrenoid matrix of the siphonous green alga, Caulerpa okamurae Web.v.Bos.

The specific localization and unique shape of the chloroplast nucleoid(ct-nucleoid)in the pyrenoid of C. okamurae Web. v. Bos. were revealed by fluorescence microscopy after staining with the DNA specific fluorochrome 4'6-diamidino-2-phenylindole(DAPI).

Disappearance of Chloroplast Nucleoids during Male Gamete Formation in Bryopsis plumosa (Hudson) C. Ag. (Chlorophyceae)

Changes in chloroplast nucleoids accompanying chloroplast degeneration during male gamete formation in the coenocytic green alga, Bryopsis plumosa, was observed by fluorescence microscopy using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). The fusiform chloroplasts of vegetative cells always included numerous nucleoids, which seemed to be scattered at random throughout the chloroplast except in the pyrenoid. When gamete formation was initiated, all chloroplasts in the male gametangia turned yellowish green. At this stage, each chloroplast assumed a moniliform shape after constriction at several sites and contained many nucleoids. Subsequently, the chloroplast divided into smaller segments, each of which was then apportioned to a young male gamete. The internal structure of the chloroplast degenerated considerably with time. Nucleoids could not be discerned with DAPI staining in chloroplasts of either young or mature male gametes, but occasionally fluorescent areas could be seen within the cytoplasm of the gametes. In contrast, many nucleoids were always observed in the female gamete chloroplast. This study indicates that chloroplast nucleoids disappear during an early stage of male gamete formation. However, chloroplast DNA may be retained at a level beyond the resolution of the detection method.

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Root meristems of Pisum sativum L. cv. Lincoln were pulsed with bromodeoxyuridine, and after partial DNA denaturation the incorporated precursor was detected with an indirect immunofluorescent method by means of commercial anti‐bromodeoxyuridine monoclonal antibody and fluorescein isothiocyanate‐labeled secondary antibody. The nuclei were counterstained with the DNA‐specific fluorochrome 4′, 6‐diamidino‐2‐phenylindole (DAPI). The labeling indices (percentage of labeled calls) determined on slides prepared with the same nuclear population were very similar and in strict accordance with autoradiographic data. When DAPI fluorescence of the nuclei was measured, the unlabeled nuclei coincided with the G1 and part of the G2 regions, whereas all the labeled nuclei were in the S or G2 region. The method is reliable and, in contrast to autoradiography, data can be collected rapidly and almost immediately after the pulse. Furthermore, labeling index and the distribution of DNA contents can be obtained from the same side.

Targeting of chemical mutagens to differentiating B-lymphocytes in vivo: detection by direct DNA labeling and sister chromatid exchange induction.

In vivo systems for analyzing mutagen interactions with a specific differentiating cell population are rare. Taking advantage of the unique anatomical features of the bursa of Fabricius in the chicken, we explored the possibility of targeting chemical mutagens to a defined differentiating cell population in the animal, namely, the B-lymphocytes series. Such cells are known to be the targets for the oncogene-activating avian leukosis virus. Targeting of chemicals to cells of the bursa was demonstrated by application of the DNA-specific fluorochrome 4'-6-diamidino-2-phenylindole (DAPI) to the anal lips of neonatal chicks. Bright nuclear fluorescence of cells in the bursa was demonstrated to occur within minutes after the application of 500 microliters of DAPI. DAPI labeling of nuclei was detected up to several days after a single application. No nuclear labeling was exhibited in cells of neighboring tissues. Methyl methanesulfonate (MMS) (10 microliters) was applied to the anal lips of day-old chicks to study dose-response kinetics for mutagen targeting to DNA of dividing B-lymphocytes in the bursa. Since the mitotic index was found to be quite high (25-30%) in the bursa, chromosome analysis was used to assay for genome damage. Sister chromatid exchange frequencies of 3.9, 7.3, and 9.0 (baseline 2.5) per cell were obtained at MMS dosages per animal of 50 micrograms, 100 micrograms, and 200 micrograms respectively. These indicate the rapid and quantitative localization of DNA-binding chemicals to cells of the bursa, particularly the resident B-lymphocytes. The bursa should be a useful system for studying mutagen-DNA interactions in the differentiating B-lymphocyte and subsequent influences on the development of immunity and lymphoproliferative disease.

THE ROLE OF SECONDARY PIT CONNECTIONS IN RED ALGAL PARASITISM1

ABSTRACTSecondary pit connections are common between cells of hosts and parasites in the widespread phenomenon of red algal parasitism. The DNA‐specific fluorochrome 4′,‐6‐diamidino‐2‐phenylindole (DAPI) reveals that in host‐parasite secondary pit connection (SPC) formation between the parasitic red algaChoreocolax polysiphoniaeand its hostPolysiphonia confusa,a nucleus and other cytoplasmic components of the parasite are delivered into the cytoplasm of a host cell. Host cells receive large numbers of parasite nuclei and these, apparently arrested in G1, are maintained intact in host cells for periods of several weeks. Within these enlarged, differentiated cells, starch accumulates and cytoplasmic organelles proliferate as the central vacuole decreases in size. Host nuclear DNA synthesis is stimulated in the infected host cell, resulting in an increase in the number of host nuclei, or an increase in DNA in each of the existing host nuclei (i.e. somatic polyploidy). Occasionally, infected host cells will recommence division and engender a new host branch. Microspectrofluorometry of nuclear DNA quantitatively confirms not only the identity and transfer of parasite nuclei to host cells, but also the transfer of parasite nuclei to other parasite cells. Measurements also reveal that the single nucleus ofChoreocolaxbecomes progressively more polyploid as cells become larger and more highly differentiated. Secondary pit connection formation betweenChoreocolaxandPolysiphoniaprovides the mechanism for the transfer of parasite genetic information (via the parasite nucleus and cytoplasm) into the host. The parasite nuclei may thereby control and redirect the physiology of the host for the benefit of the parasite.

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).

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.

Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.