DAPI: a DNA-Specific Fluorescent Probe

DAPI (4′,6-diamidino-2-phenylin-dole) is a DNA-specific probe which forms a fluorescent complex by attaching in the minor grove of A-T rich sequences of DNA. It also forms nonfluorescent intercalative complexes with double-stranded nucleic acids. The physicochemical properties of the dye and its complexes with nucleic acids and history of the development of this dye as a biological stain are described. The application of DAPI as a DNA-specific probe for flow cytometry, chromosome staining, DNA visualization and quantitation in histochemistry and biochemistry is reviewed. The mechanisms of DAPI-nucleic acid complex formation including minor groove binding, intercalation and condensation are discussed.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4',6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

DAPI as a Useful Stain for Nuclear Quantitation

A simple-to-use fluorescent stain, 4′,6-diamidino-2-phenylindole (DAPI), visualizes nuclear DNA in both living and fixed cells. DAPI staining was used to determine the number of nuclei and to assess gross cell morphology. Following light microscopic analyses, the stained cells were processed for electron microscopy. Cells stained with DAPI showed no ultrastructural changes compared to the appearance of cells not stained with DAPI. DAPI staining allows multiple use of cells eliminating the need for duplicate samples.     

Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique

The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.     

Flow Cytometric Applications of Sulforhodamine 101 as a Fluorescent Stain for Total Cellular Protein

In this paper, the use of Sulforhodamine 101 (SR 101; C.I. 14318) as a fluorescent stain for flow cytometric determinations of total cellular protein (TCP) is described. Flow cytometric quantification of TCP fluorescence can provide a valuable analytical parameter for assessing both changes occurring in overall cellular protein content, such as in response to blast transformation, and heterogeneity in cellular size within a specimen, such as a tumor. Very little information is available in the literature pertaining to the use of SR 101 as a protein stain. Like fluorescein isothiocyanate (FITC), SR 101 can be excited at 488 nm; however, it binds ionically and has an emission maximum at 600 nm, which is advantageous in certain staining and filter combinations. In this report, the utility of SR 101 staining is demonstrated using pokeweed mitogen-stimu-lated lymphocytes and cycloheximide- and di-methylsufloxide-treated cells. Single, two- and three-color flow cytometric applications are possible, using SR 101 in combination with 4',6-diamidino-2-phenylindole (DAPI) and/or FITC.     

一种新型的DNA荧光染料—DAPI的光学特性及其应用

近年来,由于荧光显微镜的改进,荧光染料的不断增多以及免疫荧光技术的发展,荧光显微术已在细胞生物学的研究中广泛应用。荧光显微术用于研究细胞核和染色体已有较长的历史,不过目前除了哺乳类染色体分带荧光技术和孚尔根型荧光染色有所发展外,曾经使用过的一些核的荧光染色方法已应用不多。     

一种新型的DNA荧光染料-DAPI的光学特性及其应用

近年来,由于荧光显微镜的改进,荧光染料的不断增多以及免疫荧光技术的发展,荧光显微术已在细胞生物学的研究中广泛应用。荧光显微术用于研究细胞核和染色体已有较长的历史,不过目前除了哺乳类染色体分带荧光技术和孚尔根型荧光染色有所发展外,曾经使用过的一些核的荧光染色方法已应用不多。最近,一种新型的DNA荧光染     

An excision and squash technique for analysis of the cell cycle in the root quiescent centre of maize

The quiescent centre of primary roots of Zea mays L. (cvs LG 11 and Golden Bantam) consists of a population of slowly cycling diploid cells. These metabolically inactive cells may be triggered to synthesise DNA under specific conditions and constitute a good model for studying the regulation of the cell cycle. An excision and squash technique is described for the quiescent centre which, when coupled with Feulgen and fluorochrome staining, allows nuclear DNA contents to be determined by microdensitometry in less than a day. This technique was coupled with experiments in which excised quiescent centres were placed on solid culture medium into which hormones and radioactive DNA precursors were incorporated. In complementary and control experiments [methyl-³H]thymidine was supplied to intact roots (with or without root caps) by means of fibre-glass cubes as donors.
Progression of the cell cycle was followed by microdensitometry and autoradiography. Distribution of DNA content was similar in excised and squashed quiescent centres and in histological sections. Labelling experiments showed that the quiescent centre is made up of cells that differ in their cycle time. While some labelled cells had reached mitosis after 8 h, others were still in G₂ after 16 h of continuous labelling. Excision and culture of the quiescent centre resulted in a dramatic activation of the cell cycle as shown by the labelling index that increased from 15% in intact roots fed during 16 h with [methyl-³H]thymidine to 31% in excised quiescent centres to which radioactive precursor was supplied during the same time. Supplying hormones (50 μ M abscisic acid [ABA], 0. 1 μ M zeatin, 1 μ M zeatin riboside) to quiescent centres via the culture medium restored their inactivity (labelling indices dropped to 1% after ABA. and to 11% after zeatin and zeatin riboside treatments. respectively).     

Comparison of ethidium bromide and 4′,6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels

Two methods of quantitative, fluorescent detection of DNA bands in agarose gels separated by electrophoresis were evaluated for sensitivity and linearity of response. Comparisons of ethidium bromide staining with a method using 4′,6-diamidino-2-phenylindole (DAPI) developed in this work showed that DAPI is several times more sensitive than ethidium bromide in conditions of comparable background flourescence. Optimum flourescent staining and detection conditions for DNA bands in agarose gels using DAPI are desctribed, and advantages of this method over other fluorescent detection methods are discussed.     

Flow cytometric assessment of cell viability: a multifaceted analysis

Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.     

DNA affinity binding studies using a fluorescent dye displacement technique: the dichotomy of the binding site

We have observed a number of discrepancies and contradictions in the use of a fluorescent intercalator displacement assay in surveying the binding affinities of dinuclear polypyridyl ruthenium(II) complexes with DNA. By a modification of the assay using the fluorescent minor-groove binder 4′,6-diamidino-2-phenylindole, rather than intercalating dyes (ethidium bromide or thiazole orange), results were obtained for all complexes studied which were consistent with relative affinities and stereoselectivities observed with other techniques, including NMR, affinity chromatography and equilibrium dialysis. It is believed that the difference in binding mode between the minor groove-binding Ru(II) complexes and the intercalating fluorescent dyes they are displacing may contribute to these discrepancies. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

De novo origin of multiple small supernumerary marker chromosomes (sSMCs) in a child with intellectual disability and dysmorphic features

Small supernumerary marker chromosomes (sSMCs) are a heterogeneous group with regards to their clinical effects as well as their chromosomal origin and their shape. The sSMCs are associated with mental retardation and dysmorphic features. Multiple sSMCs are rarely reported. We report four sSMCs in a case of dysmorphic features and intellectual disabilities. Among the four sSMCs, one sSMC confirmed to be chromosome 5 derived sSMC using fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). The sSMCs were de novo originated as parental chromosomal analysis revealed normal karyotypes. The sSMC derived from chromosome 5 might be associated with mental retardation and dysmorphic features in the present case. However the remaining three sSMCs might have originated from repetitive sequences of chromosomes.     

First Experimental Evidence for the Presence of a CRISPR Toxin in Sulfolobus

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.     

Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water

AIMS: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment. METHODS AND RESULTS: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed. This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens. An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed. This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r2 = 0.96 and 0.93, respectively). CONCLUSION: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time. This system could be easily applied to the rapid analysis of microbes in various environments.     

Peanut witches' broom (PnWB) phytoplasma-mediated leafy flower symptoms and abnormal vascular bundles development

The peanut witches'' broom (PnWB) phytoplasma causes virescence symptoms such as phyllody (leafy flower) in infected peanuts. However, the obligate nature of phytoplasma limits the study of host-pathogen interactions, and the detailed anatomy of PnWB-infected plants has yet to be reported. Here, we demonstrate that 4′,6′-diamidino-2-phenylindole (DAPI) staining can be used to track PnWB infection. The DAPI-stained phytoplasma cells were observed in phloem/internal phloem tissues, and changes in vascular bundle morphology, including increasing pith rays and thinner cell walls in the xylem, were found. We also discerned the cell types comprising PnWB in infected sieve tube members. These results suggest that the presence of PnWB in phloem tissue facilitates the transmission of phytoplasma via sap-feeding insect vectors. In addition, PnWB in sieve tube members and changes in vascular bundle morphology might strongly promote the ability of phytoplasmas to assimilate nutrients. These data will help further an understanding of the obligate life cycle and host-pathogen interactions of phytoplasma.     

Destruction of organelle nuclei during spermatogenesis inChara corallina examined by staining with DAPI and anti-DNA antibody

Summary The behavior of plastid and mitochondrial nuclei (synonymous with nucleoids) during spermatogenesis inChara corallina was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI). These organelle nuclei, which were present in internode cells and cells at the early spermatid stage, disappeared during spermatogenesis. This conclusion was confirmed by immunofluorescence microscopy using of a monoclonal anti-DNA antibody. The pattern of fluorescence obtained using the antibody coincided with that obtained by staining with DAPI. These results suggest that the disappearance of nuclei from male-derived organelles is most likely to result in maternal inheritance inChara corallina.     

Trashing of Single-Stranded DNA Generated during Processing of Arrested Replication Fork in E. coli

We analyzed formation of single-stranded DNA (ssDNA) related to SOS induction in nalidixilate (Nal)-treated Escherichia coli, using immunofluorescence microscopy accompanied by computer analysis. We found enhancement of both ssDNA concentrations and cells having ssDNA foci that often localized around cellpoles. Analyzing several mutants deficient in DNA repair or replication, we found, after Nal treatment, that recN, recA, uvrD and dnaB failed to increase ssDNA concentration and that recG and particularly ruvA only partially enhanced it. In Nal-treated recB mutant, despite its failure in SOS induction, ssDNA foci positive cells increased with a slight enhancement of its concentration. These observations suggest the existence of a cellular process that sequesters genotoxic ssDNA as inert form, offering a new concept for SOS suppressor genes action.     

Dynamic Regulatory Interactions of Rad51, Rad52, and Replication Protein-A in Recombination Intermediates

Rad51, Rad52, and replication protein-A (RPA) play crucial roles in the repair of DNA double-strand breaks in Saccharomyces cerevisiae. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 recruits Rad51 into single-stranded DNAs (ssDNAs) that are saturated with RPA. Rad52 also promotes annealing of ssDNA strands that are complexed with RPA. Specific protein-protein interactions are involved in these reactions. Here we report new biochemical characteristics of these protein interactions. First, Rad52-RPA interaction requires multiple molecules of RPA to be associated with ssDNA, suggesting that multiple contacts between the Rad52 ring and RPA-ssDNA filament are needed for stable binding. Second, RPA-t11, which is a recombination-deficient mutant of RPA, displays a defect in interacting with Rad52 in the presence of salt above 50 mM, explaining the defect in Rad52-mediated ssDNA annealing in the presence of this mutation. Third, ssDNA annealing promoted by Rad52 is preceded by aggregation of multiple RPA-ssDNA complexes with Rad52, and Rad51 inhibits this aggregation. These results suggest a regulatory role for Rad51 that suppresses ssDNA annealing and facilitates DNA strand invasion. Finally, the Rad51-double-stranded DNA complex disrupts Rad52-RPA interaction in ssDNA and titrates Rad52 from RPA. This suggests an additional regulatory role for Rad51 following DNA strand invasion, where Rad51-double-stranded DNA may inhibit illegitimate second-end capture to ensure the error-free repair of a DNA double-strand break.