Location of B- and Z-DNA in the chromosomes of a primitive eukaryote dinoflagellate.
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The usual conformation of DNA is a right-handed double helix (B-DNA). DNA with stretches of alternating purine-pyrimidine (G-C or A-T) can form a left-handed helix (Z-DNA). The transition B----Z, facilitated by the presence of divalent cations, cytosine methylation, or constraints on DNA such as superhelicity may play a role in the regulation of gene expression and/or in DNA compaction (Zarling, D. A., D. J. Arndt-Jovin, M. Robert-Nicoud, L. P. McIntosh, R. Tomae, and T. M. Jovin. 1984. J. Mol. Biol. 176:369-415). Divalent cations are also important in the structure of the quasi-permanently condensed chromosomes of dinoflagellate protists (Herzog, M., and M.-O. Soyer. 1983. Eur. J. Cell Biol. 30:33-41) which also have superhelicity in their DNA. The absence of histones in dinoflagellate chromosomes suggest that the search for Z-DNA sequences might be fruitful and could provide one indication of the physiological role of this particular DNA conformation. We report a complete immunofluorescent and immunogold analysis of the nuclei of the dinoflagellate Prorocentrum micans E. using monoclonal and polyclonal anti-B and anti-Z-DNA antibodies. Positive labeling was obtained with immunofluorescence using squash preparations and cryosections, both of which showed the intranuclear presence of the two DNA conformations. In ultrathin sections of aldehyde-prefixed, osmium-fixed, and epoxy-embedded cells, we have localized B-DNA and Z-DNA either with single or double immunolabeling using IgG labeled with 5- and 7-nm gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their arch-shaped configuration, are abundantly labeled with anti-B-DNA antibody. Extrachromosomal anti-B-DNA labeling is also detected on the nucleoplasm that corresponds to DNA loops; we confirm the presence of these loops previously described external to the chromosomes (Soyer, M.-O., and O. K. Haapala. 1974. Chromosoma (Berl.). 47:179-192). B labeling is also visible in the nucleolus organizer region (NOR) and in the fibrillo-granular area (containing transcribing rDNA) of the nucleolus. Z-DNA was localized in limited areas inside the chromosomes, often at the periphery and near the segregation fork of dividing chromosomes. In the nucleolus, Z-DNA is observed only in the NOR area and never in the fibrillo-granular area. For both types of antibody experiments, controls using gold-labeled IgG without primary antibody were negative. A quantitative evaluation of the distribution of the gold-labeled IgG and a parametric test support the validity of these experiments.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)
Distortions induced in double-stranded oligonucleotides by the binding of cis- or trans-diammine-dichloroplatinum(II) to the d(GTG) sequence.
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Conformational changes induced in double-stranded oligonucleotides by the binding of trans- or cis-diamminedichloro platinum(II) to the d(GTG) sequence have been characterized by means of melting temperatures, electrophoretic migrations in non-denaturing polyacrylamide gels, reactivities with the artificial nuclease Phenanthroline-copper and with chemical probes. The cis-platinum adduct behaves more as a centre of directed bend than as a hinge joint, the induced bend angle being of the order of 25-30 degrees. The double helix is locally denatured over 2 base pairs (corresponding to the platinated 5'G residue and the central T residue) and is distorted over 4-5 base pairs. The trans-platinum adduct behaves also more as a centre of directed bend than as a hinge joint, the induced bend angle being of the order of 60 degrees. The double helix is locally denatured over 4 base pairs (corresponding to the immediately 5'T residue adjacent to the adduct and to the three base residues of the adduct). Both the cis- and trans-platinum adducts decrease the thermal stability of the double helix. (+info)
Plant tissues in 3D via X-ray tomography: simple contrasting methods allow high resolution imaging.
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Sensory innervation of gingival and alveolar mucosa of the house musk shrew (Suncus murinus).
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The lingual gingival and the alveolar mucosa of mandible of the house musk shrew (Suncus murinus) were stained by methylene blue vital staining or osmic acid staining, and mounted as whole thickness preparations. The sensory innervation and the distribution of sensory receptors were investigated with a light microscope. The nerve fibers supplying these regions derive from the sublingual nerve, which ascend in the mucosa as they branch out. Sensory receptors found in the present study are of four kinds; free nerve endings, bush-like nerve endings, Merkel cell-neurite complexes and encapsulated corpuscles. The Merkel cell-neurite complexes were scarce and localized in the upper margin of gingival mucosa. The bush-like nerve endings were distributed preferentially in the alveolar mucosa, in which their maximum density was 9-23 per mm2. Among the organized receptors, the encapsulated corpuscles appeared most frequently throughout the mucosal area investigated, and their maximum density amounted to 27-56 per mm2 in the gingival mucosa. These corpuscles were relatively small and poorly differentiated. Although the bush-like nerve endings and the encapsulated corpuscles were fewer in the third molar region, there was no obvious regional difference in their distribution densities from the premolar region to the second molar region. (+info)
A rapid method for staining large chylomicrons.
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In this report, we present a rapid method for producing high-quality micrographs suitable for determining the size distributions of particles in concentrated samples of postprandial chylomicrons and chylomicron remnants. The procedure consists of mixing particles with osmium tetroxide in water to stabilize the lipids of the particles. These fixed and positively stained particles are then negatively stained with phosphotungstate in the presence of dilute sucrose. This dual staining procedure prevents the fusion and clustering of chylomicrons during processing for electron microscopy and is effective with particles of different lipid compositions. In addition, this procedure is simple and rapid, adding only one mixing step and 5 min to the preparation time required for conventional negative stains. (+info)
Slight changes in conditions influence the family of non-B-DNA conformations of the herpes simplex virus type 1 DR2 repeats.
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The segment inversion site of herpes simplex virus type 1 contains a series of tandem repeats with a purine bias on one strand and high G + C content (DR2 repeats) capable of adopting a non-B-DNA structure under a variety of conditions. Plasmids carrying eight contiguous copies of DR2 sequences undergo a series of supercoil-driven conformational transitions resulting in different extents of relaxation at pH 5.0. These transitions depend on the presence of an appropriate concentration of divalent cations (Mg2+ and Ca2+) which seem to interact specifically with the alternate structure(s). The transitions occurred at approximately the same superhelical density for all lengths of inserts studied. However, the onset of the transition can be shifted to lower negative superhelical densities by increasing NaCl concentrations. This leads to a reduction of the cooperativity of the transition, which takes place over a range of linking isomers under these conditions. Extrapolating from these results, we established physiological conditions where the alternate DNA structure is found at negative superhelical densities as low as -0.035. The existence of non-B-DNA conformations and/or the structural transitions of these sequences located in this region of intense biological activity implies their involvement in the life cycle of the virus. (+info)
Echinomycin-induced hypersensitivity to osmium tetroxide of DNA fragments incapable of forming Hoogsteen base pairs.
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To show conclusively that the critical structural deformation of double-helical DNA that is induced by the binding of quinoxaline antibiotics does not involve the formation of Hoogsteen base pairs, we have prepared a DNA fragment containing the nucleoside analog 7-deaza-2'-deoxyadenosine in one of the two strands. This DNA fragment was subjected to treatment with the thymidine-specific reagent osmium tetroxide and to DNase I "footprinting" in the presence or absence of micromolar concentrations of echinomycin. We report that this anti-tumor antibiotic binds to DNA containing the nucleoside analog as well as to natural DNA and that the previously reported hypersensitivity to osmium tetroxide of certain thymidine residues adjacent to echinomycin binding sites is maintained in analog-containing DNA. Since these thymidines are rendered incapable of participating in Hoogsteen base pairs by the incorporation of 7-deaza-2'-deoxyadenosine, we conclude that this unusual base-pairing scheme is not the cause of the observed hypersensitivity to osmium tetroxide and that it therefore results from a large local unwinding of the DNA in the presence of the antibiotic. Moreover, preventing the possibility of Hoogsteen base pairing does not preclude echinomycin binding. (+info)
Complex structural behavior of oligopurine-oligopyrimidine sequence cloned within the supercoiled plasmid.
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Synthetic sequence GATCC(AG)7ATCG(AT)4CG(AG)7 was cloned into plasmid and its structural behavior under the influence of supercoiling was analysed by chemical modification at variety of experimental conditions. It was found that this sequence adopts at least two different non-B conformations depending on -delta and pH values. Moreover, 12 nucleotide long non-pur.pyr spacer region separating two identical (AG)7 blocks does not provide a significant energy barrier protecting against unusual structures formation. (+info)