Application of pyronin Y(G) in cytochemistry of nucleic acids

Abstract

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y (PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation.The products of PY interaction with doublestranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry.PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 pM, the RNA stainability decreases, perhapsIn an accompanying paper (171, we describe biophysical and biochemical studies on pyronin Y (PY) and interactions of this dye with natural and synthetic nucleic acids in solutions. Several observations from these studies could be of importance for the qualitative and quantitative application of PY, both as an absorption dye for light microscopy (2,191 and as a fluorochrome (4,23,26,28). In the present paper, we demonstrate that the biophysical properties of the dye complexes with nucleic acids observed in solutions (17) are relevant in cytochemistry. The combined biophysical and cytochemical approach as described in this and the accompanying paper (17) makes it possible to better understand the complexity of the dye-nucleic acid interactions and define optimal conditions for use of this dye as a specific cytochemical probe of nucleic acids. MATERIALS AND METHODSChinese hamster cells (CHO, originally obtained from Dr. T.T. Puck) were maintained in exponential growth due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in 6 2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.Key terms: RNA content, RNA conformation, nuclear RNA, flow cytometry, CHO cells, mitosis phase either as monolayer cultures, or in suspension culture, free of mycoplasma contamination, in Ham's F-10 medium supplemented with 10% heat-inactivated newborn calf serum, as previously described (6). To obtain populations enriched in mitotic cells, the suspension cultures were treated with 0.1 pg/ml Colcemid (GIBCO, Grand Island, NY) for up to 3 hr; this resulted in a n...

Interactions of the QacR Multidrug-Binding Protein with Structurally Diverse Ligands: Implications for the Evolution of the Binding Pocket

“…For example, the ability of a given ligand to enhance qacA transcription is related not only to how strongly it is bound by QacR but also to the relative affinity of the compound for its target site, the ease with which it gains entry into the cytoplasm, and the presence of MDR transporter homologues which may export the compound in question, thereby reducing its intracellular concentration. For the specific case of ethidium, proflavin, and pyronin Y, the discrepancy between their relatively low K d values, ranging from 0.81 to 5.15 µM, but only weak induction of P qacA in vivo (Table 2) may be related to the ability of these compounds to intercalate DNA (39). This property could result in a reduction in the effective intracellular concentrations of such QacR ligands due to their nonspecific sequestration in chromosomal DNA.…”

A novel human B cell subpopulation representing the initial germinal center population to express AID

“…18 Briefly, the isolated B-cell populations were fixed in 70% ethanol overnight, then resuspended in a solution of 2 g/mL Hst (Molecular Probes, Eugene, OR) and 4 g/mL PY (Polysciences, Warrington, PA) and measured by flow cytometry on a MoFlo equipped with UV laser (DakoCytomation). Since RNA staining with PY yields a continuous histogram without demarcation between positive and negative cells, 19 an arbitrary analysis window comprising about 90% of Bm1 and Bm2 subpopulations displaying minimal PY staining was used to designate the G 0 fraction in all experiments.…”

“…Consistent with their CD71 expression, an analysis of DNA/RNA content revealed that a majority of pro-GC tonsil cells are in a G 1 state at any given time. They were then fixed and stained with pyronin Y and Hoechst 33342 dye [18][19][20] (Figure 3). To our knowledge, this technique has not been previously reported for tonsillar B-cell subpopulations.…”

New double-staining technique for RNA and DNA measurement in marine phytoplankton

“…PY had been reported to specifically stain RNA in combination with DNA-specific stains, namely H342 (Tanke et al 1980, Shapiro 1981. Nevertheless, the considerable fluorescence-quenching of PY by increasing amounts of RNA added (due to the guanine bases, according to Darzynkiewicz et al 1987 andKapuscinski &Darzynkiewicz 1987) was a major limitation. The main interest in A 0 was the possibility of distinguishing between DNA and RNA based on the metachromatic properties of the dye (Darzynluewicz 1979, Darzynkiewicz et al 1983.…”