Elsevier

Acta Histochemica

Volume 106, Issue 6, 24 February 2005, Pages 413-420
Acta Histochemica

DNA-binding fluorochromes: correlation between C-banding of mouse metaphase chromosomes and hydrogen bonding to adenine–thymine base pairs

https://doi.org/10.1016/j.acthis.2004.09.001Get rights and content

Summary

Numerous non-rigid and bow-shaped cationic fluorochromes exist which bind to chromatin DNA, but only some of them are able to selectively label pericentromeric heterochromatin (C-bands) in metaphase chromosomes. Likewise, some DNA ligands allow the recognition of adenine–thymine sequences establishing hydrogen bonds with acceptor atoms in the minor groove. In the present study, we have employed cationic fluorochromes based on a variety of chemical groups to analyze comparatively their potential to demonstrate C-bands, as well as the relationship between this feature and structural parameters of fluorochromes such as curvature radius and hydrogen-bonding ability. Only fluorochromes that bind DNA by hydrogen bonds demonstrated the characteristic C-banding pattern in mouse metaphase chromosomes (together with a weak G/Q-banding), whereas the other (non-hydrogen-bonding) cationic fluorochromes produced uniform emission without any visible banding.

Introduction

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 (Sumner, 1990; Stockert et al., 1990; Bella and Gosálvez, 1994; Bella et al., 1995; Bickmore and Craig, 1997; Pinna-Senn et al., 2000; Haugland, 2002). 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 (Stokke and Steen, 1985; Kopka and Larsen, 1992; Burckhardt et al., 1993; Krugh, 1994; Kahne, 1995; Geierstanger and Wemmer, 1995; Horobin and Kiernan, 2002).

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 (Goodsell and Dickerson, 1986; Zasedatelev, 1991), and some minor groove ligands have been shown to recognize specific sequences in DNA (Kopka and Larsen, 1992; Dwyer et al., 1993; White et al., 1998).

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 (e.g. thioflavine T for amyloid; see Horobin and Kiernan, 2002), and in microscopical studies of chromatin (Latt et al., 1984; Stockert et al., 1990, Stockert et al., 1991, Stockert et al., 1997b). 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 (Hörz and Altenburger, 1981), which contains the EcoRI GAATTC restriction site and numerous consecutive adenines ([dA]4−6–[dT]4−6), sometimes flanked by thymines (Redi et al., 1990). 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.

Section snippets

Metaphase chromosome preparations

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.

Fluorochromes

Preparations were stained with the following fluorochromes: Hoechst 33258 (Sigma, St.

Results

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 (Fig. 1A) produced C-bands in mouse metaphase chromosomes (Fig. 2B), whereas fluorochromes lacking H-bonding capacity (Fig. 1B) did not generate C-bands (Fig. 2A). In order to enhance the contrast differences between C-bands and chromosome arms, the original color images of metaphase chromosomes

Discussion

In addition to the widely used fluorochromes such as DAPI and H-33258, other cationic compounds also interact with DNA but their binding mechanism and base pair specificity are not well known. The fluorochromes used here correspond to a wide collection of chemical groups such as benzimidazole (Hoechst 33258, nuclear yellow), aromatic diamidine and analogues (DAPI, berenil, 2-hydroxystilbamidine, M&B 938, D-288/45, D-288/48), diphenylmethane (auramine O), polymethine (pinacyanol, pyrvinium, DiOC1

Acknowledgements

We thank M.I. Ortiz, G. Dalmasso, J. Gosálvez and G.R. Solarz for valuable collaboration. This study was supported by grants SAF2002-04034-C02-01, BOS 2002-00232 and BOS 2003-04263 (Spain), and SECyT-18/C070, Universidad Nacional de Río Cuarto (Argentina). JCS is a scientific member of the Consejo Superior de Investigaciones Científicas (CSIC), Spain.

References (41)

  • J.L. Bella et al.

    C-banding plus fluorochrome staining shows differences in C-, G-, and R-bands in human and mouse metaphase chromosomes

    Genome

    (1995)
  • W. Bickmore et al.

    Chromosome bandspatterns in the genome

    (1997)
  • G. Burckhardt et al.

    Mechanisms of distamycin A/DAPI chromosome staining. I. Competition binding effects of nonintercalative DNA groove-binding agents in situ and in vitro

    Cytogenet Cell Genet

    (1993)
  • T. Cuéllar et al.

    Fluram induces species-dependent C and G bands in mammalian chromosomes, revealing heterogeneous distribution of chromosomal proteins

    Genome

    (1991)
  • T.J. Dwyer et al.

    Structural analysis of covalent peptide dimers, bis(pyridine-2-carboxamidonetropsin) (CH2)3−6, in complex with 5′-TGACT-3′ sites by two-dimensional NMR

    J Am Chem Soc

    (1993)
  • J. Espada et al.

    Fluorescence of mast cell granules in paraffin sections and cell smears induced by an N-quaternary oxazole scintillator

    Histochem J

    (1995)
  • L. Ferrucci et al.

    A cytological approach to the role of guanine in determining quinacrine fluorescence response in eukaryotic chromosomes

    J Histochem Cytochem

    (1982)
  • K. Fukui et al.

    Somatic chromosome map of rice by imaging methods

    Theor Appl Genet

    (1991)
  • H. Geierstanger et al.

    Complexes of the minor groove of DNA

    Annu Rev Biophys Biomol Struct

    (1995)
  • D. Goodsell et al.

    Isohelical analysis of DNA groove-binding drugs

    J Med Chem

    (1986)

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