Absence of nucleosomes in a histone-containing nucleoprotein complex obtained by dissociation of purified SV40 virions.

Reduction of disulfide bonds involving the major capsid protein with dithiothreitol and removal of the calcium ions by EGTA disrupts the simian virus 40 virions. This process yields normal circular viral minichromosomes containing the four core histones and traces of the capsid proteins at pH values higher than 8.5. However, when carried out at pH 7.5, this procedure yields nucleoprotein complexes that contain both histones and the viral structural proteins. These pH 7.5 complexes appear as circular structures with a mean of 93 +/- 17 beads with a diameter of 7 nm and no visible nucleosomes when observed by electron microscopy. In contrast to the compaction of the viral DNA in minichromosomes, the length of these beaded structures is roughly the same as free DNA. We suggest that VP1, the major capsid protein, can act as a nucleosome unfolding agent in neutral pH and low ionic strength.

[O'Brien's actinic granuloma, Three cases (author's transl)]. / Le granulome actinique de O'Brien. A propos de 3 observations.

We report three cases of 54, 58 and 65-year-old patients presenting annular lesions with centrifugal migratory extension. They occur exclusively on sun-exposed areas. In two cases, the lesions were multiple. In the third case, the lesion was single and mimicked an erythema annulare centrifugum. The past history ranged from 18 months to 8 years. Microscopic examination of the central part showed a disappearance of elastic fibers in upper reticular dermis. Examination of the ring showed in upper reticular dermis an histiocytic granulomatous infiltration with many giant cells, lymphocytes and patterns of elastic fibers phagocytosis. Similar features were found by electronmicroscopy. These three cases illustrate the typical features of O'Brien's actinic granuloma. Relationship between this actinic granuloma and granuloma annulare occurring on sun-exposed areas on one side, and necrobiosis lipoidica, Miescher's granuloma and granuloma multiforme on the other side, are discussed. On the basis of some clinical and histological patterns, the autonomy of O'Brien's actinic granuloma appears to be established.

[Chromatin structure with no nucleosomes: dissociated SV40 virus]. / Une structure chromatinienne dépourvue de nucléosomes: le virion dissocié de SV40.

The mild dissociation of SV40 virus particles at pH 7.5 yields a nucleoprotein complex containing all the capsid proteins and histones of the intact virion. Electron microscopic observations show a bead-on-a-string structure comprising about 70 particles 7 nm in diameter along the viral DNA. No particle of nucleosome size was visible. The compaction of DNA in this complex is lower than 2 and is likely to be close to unity.

Absence of nucleosomes in a fraction of SV40 chromatin between the origin of replication and the region coding for the late leader RNA.

Electron microscopic examination of SV40 chromatin prepared 44 hr post-infection led to the visualization of a nucleosome-free region (gap) in 15-20% of the minichromosomes. Minichromosomes with and without a gap displayed a mean number of 24 nucleosomes. Measurements carried out on dark field micrographs yielded for the gap a mean length of 249 +/- 13 bp, with a maximum value of 385 bp. The gap was mapped following digestion with three single-cut restriction endonucleases: Bgl l, Bam HI and Eco RI. It was located in the region of the origin of replication in accordance with previous biochemical data. To assess the situ existence of a nucleosome-free region, nuclei from infected cells were digested with DNase I. A highly sensitive region was thus revealed and mapped by secondary digestion with Eco RI. It was located in the same region as the gap, between 0.67 and 0.74 on the physical map. The sensitive region could be detected throughout the late phase of the virus cycle. These findings strongly suggest that a nucleosome-free region exists in the cells. The gap is not likely to be involved in replication, since it is asymmetric with respect to the Bgl I cleavage site, from which replication proceeds symmetrically.

Additive effects of cortisol and calf serum on RNA synthesis in isolated rat hepatocytes. Autoradiographic studies with the light and electron microscope.

The known stimulating effect of calf serum (10%) and cortisol (20 microgram/ml) on the incorporation of 2H uridine into the nuclei of isolated rat hepatocytes has been confirmed with the method of light and electron autoradiography. Moreover, it has been shown that cortisol and serum exert additive effects when they are applied jointly. In the presence of either serum or cortisol or both transcriptional activity increases gradually, reaching its maximum level after 6 h incubation. This effect is reduced when the incubation is prolonged up to 24 h. Autoradiographic ultrastructural studies have shown that both serum and cortisol enhance the nucleolar as well as extranucleolar transcriptional activity. The nuclei of hepatocytes cultivated in medium containing serum or both serum and cortisol shown a higher growth rate in comparison with those cultivated in a poor, control medium. Cortisol added to the serum-free medium does not stimulate the nuclear growth.

Methods in ultrastructural cytochemistry of the cell nucleus.

The electron microscopical study of the cell nucleus as observed in thin sections requires the use of cytochemical methods because of the intricate pattern of the nuclear components. The in situ techniques based on electron staining and enzymatic digestion are reviewed, excluding autoradiography, cytoenzymology and immunocytochemistry. A tentative classification has been adopted according to the chemical nature of the revealed component. Thus, the staining procedures for the nucleoproteins in general, for both nucleic acids, for the proteins, and finally for the deoxyribonucleoproteins and DNA are considered separately. 1--Stains for the nucleoproteins include simple reagents such as the uranyl and lead salts which are largely used in electron microscopy but are of limited specificity. 2--A variety of methods, some of them specific, is available for the simultaneous visualization of DNA and RNA which is based on common properties: basophilia, ability to bind diaminoacridines, presence of hydroxyl groups. However, due to the recent development of specific and preferential methods for each nucleic acid, we feel that among the older methods, only rapid and simple procedures for the detection of both nucleic acids remain of interest. 3--Proteins being ubiquitous, the useful techniques must reveal subsets within the total nuclear proteins. Apart from some endogeneous enzymes, basic proteins -- practically histones -- so far represent the only group for the detection of which reliable methods exist. 4--Several techniques developed recently are available for the specific detection of DNA. In favourable cases, methods derived from the Feulgen reaction allow its visualization at a molecular level. In addition, standard procedures for the preparation of mammalian cells and tissues are described. Each staining method is at least briefly discussed, but emphasis has been placed on a small number of techniques described in detail. They comprise the EDTA regressive stain for the ribonucleoproteins, several reactions of the basic proteins and the Feulgen-like osmium ammine reaction for DNA.

Late transcription and simultaneous replication of simian adenovirus 7 DNA as revealed by spreading lytically infected cell cultures.

Miller's technique of spreading DNA was applied to monkey cells productively infected with simian adenovirus 7. This permitted the visualization of cellular DNA transcription, both nucleolar and non-nucleolar, and of late transcription and replication of virus. Virus double-stranded DNA, thin fibres with very few nucleosome-like particles, were observed carrying either transcription or replication complexes. In addition, both RNP transcripts and replication forks were found on some virus duplex DNA. Virus single-stranded DNA replicative intermediates were identified on the basis of their increased thickness and contrast which results from the presence of a DNA binding protein.

Localization of simian adenovirus 7 (SA 7) transcription and replication in lytic infection. An ultracytochemical and autoradiographical study.

We have studied SA7 (simian adenovirus 7) lytic infection at the ultrastruct level. The use of cytochemical techniques which specifically stain DNA or preferentially stain ribonucleoproteins permitted the analysis of the structure of the virus-induced nuclear inclusions, and revealed presumed virus DNA before the appearance of other nuclear alterations. Correlation of these findings with high resolution autoradiography enabled the functions of virus DNA replication and transcription to be ascribed to a defined nuclear inclusion. We demonstrate that the nucleolus remains distinct from the inclusion body, contrary to the situation in other adenovirus-infected cells. The functional role of host cell chromatin and of the nucleolus are discussed.