In recent years, much attention has been given to the development and microscopic applications of DNA-selective fluorescence methods for cell and molecular biology studies, DNA cytochemistry, and chromosome banding. In this respect, several ligands have shown high affinity for the DNA minor groove at adenine–thymine (AT) sequences which are known as trypanocidal, anti-parasitic and anti-tumor drugs, vital fluorescent probes, and fluorochromes.

Ligands that bind selectively to AT sequences in the DNA minor groove have the following features: (a) cationic status, (b) non-rigid and bowed shape, and (c) hydrogen (H) bonding to acceptor N3 and O2 atoms of A and T, respectively. The design of molecules that recognize a given DNA sequence would provide a useful tool to control gene expression and a more rational basis for the design of new cytochemical and pharmacological compounds. Several geometric prerequisites of AT-binding ligands have been described, and some minor groove ligands have been shown to recognize specific sequences in DNA.

AT-binding fluorochromes such as 4?,6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 are commonly used to reveal AT-rich chromosome regions, and are, therefore, applied for cytogenetic and cytochemical analysis. Other fluorochromes are used to visualize specific tissue components, and in microscopical studies of chromatin. Although there are fluorochromes which are cationic and show an adequate curvature to fit in the convex floor of the minor groove, they do not necessarily recognize AT sequences, and thus chemical parameters which could explain their selectivity for DNA binding have to be investigated.

The pericentromeric heterochromatin DNA of mouse chromosomes (C-bands) is AT-rich (approximately 69% AT) and composed of a highly repetitive 230–240 base pair unit, which contains the EcoRI GAATTC restriction site and numerous consecutive adenines ([dA]4?6–[dT]4?6), sometimes flanked by thymines. Due to the high AT content of DNA in C-bands of mouse chromosomes, they can be used as a suitable test model to analyze the selectivity of fluorochrome binding at the microscopical level. In the present study, we have analyzed the capacity of non-rigid cationic DNA fluorochromes to demonstrate C-bands in mouse metaphase chromosomes. It was found that only those that can form H bonds are able to produce C-banding.

Routine cytogenetic preparations were obtained from the bone marrow of Balb-c mice that had been injected with 0.01% colchicine (0.25 ml) at 1.5 h before sacrifice. Cell suspensions were subjected to hypotonic treatment in 0.075 M KCl during 20 min at 37 °C. After centrifugation, cells were fixed in several changes of freshly prepared methanol–acetic acid (3:1; v/v), spread onto glass slides and air-dried routinely.

Preparations were stained with the following fluorochromes: Hoechst 33258 (Sigma, St. Louis, MO, USA), nuclear yellow (2?-(4-sulfamylphenyl)-6?-(6-[4-methyl-piperazino]-2-benzimidazolyl)-benzimidazole trichloride; Sigma), 2-hydroxystilbamidine methanesulfonate (Fluka, Buchs, Switzerland), DAPI (Sigma), DAPI-related compounds D-288/45 and D-288/48 (synthesized and kindly provided by Prof. O. Dann, Department of Applied Chemistry, University of Erlangen, Germany), berenil (diminazene aceturate; 1,3-bis(4-amidinophenyl)triazine aceturate; Sigma), M&B 938 (4,4?-diamidino-diphenylamine dihydrochloride, batch 7451; supplied by Rhône-Poulenc Rorer, Essex, UK), auramine O (Sigma), DiOC1(3) (3,3?-dimethyloxacarbocyanine iodide; Sigma), Q-dmPOPOP (N-quaternized derivative prepared from dimethyl-POPOP (Merck, Darmstadt, Germany) as described by Espada et al., 1995 J. Espada, G.E. Bertolesi, C.I. Trigoso, C. Gamallo and J.C. Stockert, Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator, Histochem J 27 (1995), pp. 318–322. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (9)Espada et al., 1995), pyrvinium pamoate (Sigma), thioflavine T (Sigma), pinacyanol bromide (Sigma), astrazone pink FG (kindly provided by Bayer, München, Germany), and PyPO (1-methyl-4-(5-phenyl-2-oxazolyl)-pyridinium p-toluene sulfonate; Aldrich, Steinheim/Albruch, Germany). Some characteristics and the chemical structure of these fluorochromes are illustrated in Table 1 and Fig. 1, respectively.

Metaphase preparations were stained in the dark at room temperature for 5 min with freshly prepared solutions of the fluorochromes in distilled water (1–20 ?g/ml; Table 1), washed and mounted in glycerol–water (1:4; v/v), or allowed to dry and then mounted in DePeX (Serva, Heidelberg, Germany). Preparations were observed and photographed under a Zeiss III (Zeiss, Oberkochen, Germany) or an Olympus BX-61 (Olympus, Hamburg, Germany) epifluorescence microscope equipped with ultraviolet (UV, 365 nm), violet-blue (436 nm), blue (450–490 nm), and green (546 nm) excitation filters. Images were captured with an Olympus DP50 CCD camera and processed using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA, USA) and PowerPoint 2000 software (Microsoft, Redmond, WA, USA).

Computer-assisted image processing and analysis methods (IPA) are useful to study and characterize metaphase chromosomes. Therefore, IPA of metaphase images was performed with the public domain Scion Image Beta 4.02 software, which allows one to compare pseudocolor chromosome maps, densitometric profiles and surface plots.

In order to examine the curvature of fluorochromes and to compare their shape and size, the corresponding molecular structures were generated with the molecular modeling software HyperChem 3.0 (Hypercube, Gainesville, FL, USA). The lowest energy structure of each fluorochrome was found by optimization using the Polak-Ribiere conjugate gradient algorithm of molecular mechanics. After modeling, the radii of both the ligand curvature (rL) and DNA curvature (rDNA=11 Å) on the minor groove floor of the oligomer (dA)10–(dT)10 were calculated, the curvature index (CI)=rL/rDNA revealing the geometrical correspondence between both curvatures. Ligands with CI values lower or higher than 1 have, respectively, a too closed or a too broad curvature to complement adequately the curvature of DNA.

Optimum concentrations, optimum excitation wavelength for each fluorochrome and the color of emitted fluorescence are indicated in Table 1. All fluorochromes with the ability to bind DNA through H bonds produced C-bands in mouse metaphase chromosomes, whereas fluorochromes lacking H-bonding capacity did not generate C-bands. In order to enhance the contrast differences between C-bands and chromosome arms, the original color images of metaphase chromosomes were first converted to gray level images and then to pseudocolor images. Densitometric profiles and surface plots (not shown) also revealed clear differences between chromosomes stained with H-bonding and non-H-bonding fluorochromes, thus confirming the fact that the former type of dye generates C-bands. In addition, bright chromatin masses representing centromeric heterochromatin in interphase nuclei were clearly demonstrated by C-banding fluorochromes.