Assignment of DNA binding sites for DAPI and bisbenzimide (Hoeschst 33258). Comparative footprinting study

University of Cambridge, Department of Pharmacology, U.K.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 03/1988; 949(2):158-68. DOI: 10.1016/0167-4781(88)90079-6
Source: PubMed


DNA binding sites for the minor groove-binding ligands DAPI (4',6-diamidine-2-phenylindole) and Hoechst 33258 (bisbenzimide) have been analysed using DNAase I and micrococcal nuclease footprinting techniques. Both drugs appear to bind to AT-rich regions containing at least four such basepairs. Hoechst 33258 seems to bind relatively poorly to nucleotide sequences containing the alternating step TpA. However, in contrast to DAPI, it can more readily accommodate the presence of guanosine residues at the end of the binding site. We compare the DNA binding sites for DAPI and Hoechst 33258 with those determined for the related minor groove-binding ligands, berenil, netropsin and distamycin A, under comparable conditions, and discuss the importance of using different footprinting probes when analysing drug-DNA interactions.

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    • "Permeabilization and blocking of the tissues in PBS-TX (1% BSA, PBS, 0.3% Triton X-100) for 1 h at 4 • C was followed by incubation in primary antibodies in PBS- TX overnight at 4 • C. The samples were washed for 2 h in several changes of PBS and incubated in secondary antibodies for 4 h at room temperature. Two different nuclear dyes were used to visualize the organization of the cell clusters as they proved to be sensitive and reliable DNA markers (Portugal and Waring, 1988). Finally, all tissues were washed for at least 2 h in several changes of PBS and mounted in Mowiol (Calbiochem). "
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    ABSTRACT: In recent years, there has been a growing interest in synthesizing studies on the structure of the nervous systems with phylogenetic questions (“neurophylogeny”). However, in the discussion on the phylogeny of Malacostraca, and the question of their closest relatives, little attention has been paid to brain morphology in the basal representatives of this taxon. To close this gap in our knowledge, this study sets out to analyze the architecture of the brain of Nebalia herbstii (Leptostraca, Phyllocarida, Malacostraca) in a neurophylogenetic context. Most phylogenetic studies consider the Phyllocarida to be the sister group to the Eumalacostraca, together forming the taxon Malacostraca, so that the brain organization of N. herbstii may possess many ancestral features. Classical histological techniques and 3D reconstruction as well as immunostaining and confocal laser-scan imaging revealed that besides taxon specific characteristics, the brain organization of N. herbstii exhibits a strong eumalacostracan affinity. The four nested optic neuropils are connected by two successive chiasms. The olfactory globular tract, connecting the olfactory lobes with second order integration centers in the lateral protocerebrum, features a contralateral connection. The olfactory lobes are composed of a radial array of spherical/spheroid glomeruli but serotonergic innervations of the olfactory glomeruli are absent. These and other architectural features are discussed in comparison to the central nervous system of Eumalacostraca, Remipedia, non-malacostracan Crustacea and Hexapoda, and are synthesized into a ground pattern of the malacostracan brain that might serve as a basis for neurophylogenetic discussion of the evolutionary relationship of these taxa.
    Zoologischer Anzeiger - A Journal of Comparative Zoology 05/2013; 252(3):319–336. DOI:10.1016/j.jcz.2012.09.003 · 1.48 Impact Factor
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    • "Kinetoplast DNA is A-T rich in comparison to trypanosomatid nuclear DNA [29-33] and we reasoned that this would allow identification of the two organelles based on their different sequence bias by using two fluorescent DNA stains with different affinity for A-T or G-C rich DNA. MGB stains, such as DAPI [23] and Hoechst [24], have a binding preference for A-T rich sequences [23,24]. BPI stains, such as ethidium bromide [25], propidium iodide (PI) and SYBR green [26] have low sequence specificity in binding. "
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    BMC Biology 01/2012; 10(1):1. DOI:10.1186/1741-7007-10-1 · 7.98 Impact Factor
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    • "To determine DNA content, fish erythrocyte mean fluorescence intensity values (I f ) were compared with those for DAPI-stained chicken erythrocytes (Kapraun and Nguyen 1994; Kapraun and Dunwoody 2002) with a known DNA content of 2.5 pg (Gregory 2007). DAPI binds by a non-intercalative mechanism to adenine and thymine rich regions of DNA which contain at least four A–T base pairs (Portugal and Waring 1988). Consequently, erythrocytes that serve as standards should have an A–T content that is similar to that in the experimental group (Coleman et al. 1985). "
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