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
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.
Available from: Christian Wirkner
- "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
Available from: Richard J Wheeler
- "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  and Hoechst , have a binding preference for A-T rich sequences [23,24]. BPI stains, such as ethidium bromide , propidium iodide (PI) and SYBR green  have low sequence specificity in binding. "
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ABSTRACT: Many trypanosomatid protozoa are important human or animal pathogens. The well defined morphology and precisely choreographed division of trypanosomatid cells makes morphological analysis a powerful tool for analyzing the effect of mutations, chemical insults and changes between lifecycle stages. High-throughput image analysis of micrographs has the potential to accelerate collection of quantitative morphological data. Trypanosomatid cells have two large DNA-containing organelles, the kinetoplast (mitochondrial DNA) and nucleus, which provide useful markers for morphometric analysis; however they need to be accurately identified and often lie in close proximity. This presents a technical challenge. Accurate identification and quantitation of the DNA content of these organelles is a central requirement of any automated analysis method.
We have developed a technique based on double staining of the DNA with a minor groove binding (4'', 6-diamidino-2-phenylindole (DAPI)) and a base pair intercalating (propidium iodide (PI) or SYBR green) fluorescent stain and color deconvolution. This allows the identification of kinetoplast and nuclear DNA in the micrograph based on whether the organelle has DNA with a more A-T or G-C rich composition. Following unambiguous identification of the kinetoplasts and nuclei the resulting images are amenable to quantitative automated analysis of kinetoplast and nucleus number and DNA content. On this foundation we have developed a demonstrative analysis tool capable of measuring kinetoplast and nucleus DNA content, size and position and cell body shape, length and width automatically.
Our approach to DNA staining and automated quantitative analysis of trypanosomatid morphology accelerated analysis of trypanosomatid protozoa. We have validated this approach using Leishmania mexicana, Crithidia fasciculata and wild-type and mutant Trypanosoma brucei. Automated analysis of T. brucei morphology was of comparable quality to manual analysis while being faster and less susceptible to experimentalist bias. The complete data set from each cell and all analysis parameters used can be recorded ensuring repeatability and allowing complete data archiving and reanalysis.
BMC Biology 01/2012; 10(1):1. DOI:10.1186/1741-7007-10-1 · 7.98 Impact Factor
Available from: Ana Gabriela Jimenez
- "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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ABSTRACT: Muscle fiber hypertrophic growth can lead to an increase in the myonuclear domain (MND), leading to greater diffusion distances within the cytoplasmic volume that each nucleus services. We tested the hypothesis that hypertrophic growth in the white muscle of fishes was associated with increases in the mean DNA content of nuclei, which may be a strategy to offset increasing diffusion constraints. DAPI-stained chicken erythrocytes standards and image analysis were used to estimate nuclear DNA content in erythrocytes and muscle fibers from 17 fish species. Mean diploid (2C) values in fish erythrocytes ranged from 0.78 to 7.2 pg. Erythrocyte 2C values were used to determine ploidy level in muscle tissue of small and large size classes of each species. Within each species, mean muscle fiber diameter was greater in the large size class than the small size class, and MND was significantly greater in larger fibers for 11 of the 17 species. Nuclear DNA content per species in muscle ranged from 2 to 64C. Fiber-size dependent increases in ploidy were observed in nine species, which is consistent with our hypothesis and indicates that endoreduplication is occurring during fiber growth. However, two species exhibited significantly lower ploidy in the larger size class, and the mechanistic basis and potential advantage of this ploidy shift is unclear. These results suggest that increases in ploidy may be a common mechanism to compensate for increases in MND associated with fiber hypertrophy in fishes, although it is likely that other factors also affect ploidy changes that occur in muscle during animal growth.
Journal of Comparative Physiology B 12/2011; 182(4):531-40. DOI:10.1007/s00360-011-0635-6 · 2.62 Impact Factor
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