A map of local curvature of the pBR322 DNA has been established by electron microscopy analysis of linearized plasmid molecules. To determine their polarity these molecules are one end labelled with an avidin‐ferritin‐biotin complex and the images are digitized. Local curvature is calculated from two mathematical treatments of the DNA trajectory and expressed in term of a mean dinucleotide wedge angle. Eight regions of curvature are distinguished. The four main regions of curvature have a high content of phased AA runs. The experimental curvature map is compared to theoretical maps of curvature obtained from four available models for DNA curvature.

Steroid hormone-receptor complexes regulate the transcription of specific genes. Recent studies of high-affinity interactions between the receptors and discrete regions of DNA, together with gene-transfer experiments, have led to the precise mapping of hormone regulatory elements. Nothing is known, however, about the mechanisms whereby DNA-bound receptors modulate gene transcription. At the start of transcription in prokaryotes two oligomeric molecules of several regulatory proteins must bind to two specific DNA sites and interact with one another to regulate the binding of RNA polymerase to DNA. Using electron microscopy to observe progesterone receptor binding to regulatory regions of uteroglobin and mouse mammary tumour virus genes, we demonstrate a similar binding between receptor oligomers at two DNA sites. DNA loops are formed when the hormone regulatory elements are at a distance from one another. Thus, in common with certain prokaryotic systems, protein-protein interactions may be important in steroid hormone regulation of gene transcription.

Abstract. The usual conformation of DNA is a righthanded 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. Mclntosh, 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 aldehydeprefixed, 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-rim gold particles, respectively. Chromosomal nucleofilaments of dividing or nondividing chromosomes, as seen in ultrathin sections in their archshaped'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 fibriUo-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. We also demonstrate the preservation of antibody activity on DNA molecules preincubated in OsO4 solutions. The role of the Z-DNA conformation as a possible site for unwinding and DNA processing in chromosomes that lack nucleosomes and that are permanently condensed is discussed. DNOFLAGELLATE protists are primitive eukaryotes, as demonstrated by ultrastructural, biochemical, and molecular biological studies (25,27...

The orientation of DNA molecules has been determined by labelling one of the molecule end with a Biotin-labelled analog of dTTP (Bio-dUTP) and then by complexing the Bio-dUTP with Avidin-Ferritin. DNAs of phi X174, pBR322 and SV40 were end labelled with Bio-dUTP and imaged by Electron Microscopy (EM). This is a rapid, general method to unambiguously determine the orientation of DNA molecules for precise mapping and quantification of DNA secondary structures or protein-DNA interaction sites using EM.

Electron microscopy offers a unique potentiality to visualize individual molecules. For the last 30 years it has been used to study the structure and the interactions of various biological macromolecules. The contribution of electron microscopy is important because of its capacity to demonstrate the existence of conformational structures such as kinks, bents, loops, etc., either on naked DNA, or on DNA associated with various proteins or ligands. Increasing interest was given to such observations when it was found that they provide a direct visualization of interacting molecules involved in DNA metabolism and gene regulation. Technical advances in the preparation of the specimens, their observation in the electron microscope, and the image processing by computers have allowed the shifting from qualitative to quantitative analysis, as illustrated by a few examples from our laboratory.

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