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Biomedical subjects

P Oudet

Publications and source records attributed to P Oudet.

At least 19 recordsLinked to original sources
PubMed1992

Rational design and synthesis of phospholipids for the two-dimensional crystallization of DNA gyrase, a key element in chromosome organization.

Properties required of lipids for two-dimensional crystallization of proteins on lipid layers at the air/water interface are discussed in terms of molecular structure. These properties are related to essential features of the overall system such as (i) the fluidity and stability of the lipid film, (ii) the affinity of the protein to be crystallized for the lipids and (iii) the accessibility of the protein to the ligand in the lipid layer as well as (iv) technical constraints of the crystallization technique. The resulting ideas were tested through the rational design and synthesis of original phospholipid structures linked to novobiocin subsequently used in the production of two-dimensional crystals of DNA gyrase (B subunit), a prokaryotic type II DNA topoisomerase.

Crystallization↗
PubMed1990

Structural study of the yeast RNA polymerase A. Electron microscopy of lipid-bound molecules and two-dimensional crystals.

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.

Lipid Bilayers↗
PubMed1990

Chromatin reconstitution on small DNA rings. III. Histone H5 dependence of DNA supercoiling in the nucleosome.

Mononucleosomes were reconstituted on small DNA rings in the presence of histone H5 and relaxed to an equilibrium using calf thymus topoisomerase I. DNA products, when compared to the equilibria observed with the same minicircles in the absence of histones, showed that a linking number reduction of 1.6 to 1.7 was associated with this reconstitution, in contrast with the 1.1 to 1.2 figure reported in our recent study of the H5-free nucleosome. Gel electrophoretic properties and electron microscopic visualization of the nucleosomes suggest a correlation between this increase and a further wrapping of the DNA around the histone core from less than 1.5 turns of the superhelix in the absence of H5, to close to two turns in its presence. Implications for DNA topology in chromatin are discussed.

Animals↗
PubMed1990

Two-dimensional crystallization of DNA gyrase B subunit on specifically designed lipid monolayers.

The B subunit of DNA gyrase formed two-dimensional crystals when bound to a specifically recognized phospholipid spread into a monolayer at the air/water interface. The especially designed lipids consisted of novobiocin coupled through the 3' or 2" hydroxyl group and a hydrophilous linker of a given length to dioleoylphosphatidic acid. Two-dimensional crystals of the gyrase B subunit are formed under physiological conditions of pH and ionic strength, with no precipitant added to the solution. Crystal diffraction extended to a 2.7 nm resolution in negative stain, with unit cell parameters a = 6.1 nm, b = 7.6 nm and gamma = 64 degrees.

Bacteria↗
PubMed1990

Electron microscopic study of yeast RNA polymerase A: analysis of single molecular images.

The structural features of the yeast DNA-dependent RNA polymerase A (I) were examined by Scanning Transmission Electron Microscopy. The enzyme was absorbed in its monomeric form and negatively stained prior to digital image acquisition at low dose. The signal to noise ratio of single particle images was improved through averaging of a large number of previously aligned and partitioned images. Six classes of images were obtained reproducibly which corresponded to different projections of the enzyme. The enzyme structure was characterized by its presence of two curved arms which defined a longitudinal cleft. By analogy with the Escherichia coli enzyme, these arms could correspond to the two large subunits A135 and A190.

Chemical Phenomena↗
PubMed1990

Polarity of binding of monoclonal antibodies to tobacco mosaic virus rods and stacked disks.

Monoclonal antibodies to tobacco mosaic virus that bind only to one end of the viral rods have been shown to recognize the surface of the protein subunit designated as the bottom, which contains the right radial and left radial alpha-helices. The specificity of the antibody binding was established by immunoelectron microscopy of complexes in which the 5' end of the RNA had been exposed at the bottom of the helical virus particle. These antibodies have been shown to bind to both ends of the stacked disk aggregate of TMV protein, which is therefore bipolar. The observations on the bipolarity of this structure are inconsistent with the presumption that stacked disks are formed by aggregation of polar two-layer disks.

Antibodies, Monoclonal↗
PubMed1989

The two DNA-binding domains of yeast transcription factor tau as observed by scanning transmission electron microscopy.

Yeast transcription factor tau interacts with the intragenic promoter of tRNA genes, binding to both the A and B block elements. Affinity-purified tau factor and tau-tDNA complexes were examined by scanning transmission electron microscopy to analyze the structural features of free and DNA bound factor. The free factor appeared as two tightly associated globular domains of roughly similar size (10 nm in diameter) and mass (approximately 300 kd). A combination of these two domains results in a mass for the factor of 510-670 kd. When tau was allowed to interact with recombinant tRNA(3Leu) genes with variable A block-B block spacing, different structures were observed. With short genes, the two globular domains were not resolved and tau appeared as a large particle covering the A and B block region. On the other hand, with genes having a larger A-B distance (53 or 74 bp), mostly dumb-bell-shaped complexes were formed with individualized factor domains bound separately to the A and B blocks. A smaller proportion of the complexes appeared to consist of a large particle bound at only one site, essentially on the B block. Mapping of the binding domains in the DNA showed a good correlation with the respective positions of the A and B promoter elements. Factor binding did not induce a noticeable DNA bending, although with extended genes apparent DNA shortening and cases of DNA looping were observed. Upon cleavage of the tRNA(3Leu) gene between the A and B blocks after or prior to complex formation, the two factor domains remained attached to the same DNA fragment (mostly the B-DNA fragment). In addition, images of protein-linked, reconstituted full-length genes were also observed. These different conformational states of the tau-tDNA complexes probably reflect the dynamic aspect of the interaction of the factor with its DNA target.

DNA Polymerase III↗
PubMed1989

Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes.

Plasmid DNA was in vitro assembled into chromatin using an S-150 extract of Xenopus laevis oocytes. By varying the assembly temperature and DNA concentration it is possible to generate fully or partially assembled molecules. The fate of the in vitro preassembled molecules injected into X. laevis oocyte nuclei and their transcriptional activity were studied. Completely reconstituted molecules underwent a rearrangement of their chromatin structure after injection and showed reduced transcriptional activity compared to protein-free DNA or partially reconstituted chromatin.

Animals↗
PubMed1988

Systematic study of phospholipids linked to a steroid derivative, spread into a monolayer at the air/water interface.

Isothermal pressure-area curves of different phospholipids linked to a cortisol derivative, spread into monolayers at the air/water interface are studied. It is shown that derivatives containing saturated lipid chains and those with unsaturated chains present quite different behaviours. With saturated derivatives, the main phase transition plateau and the stability of the fluid phase are very sensitive to the length of the lipid chains, the presence of a spacer between the lipid and the steroid moieties, the temperature and the presence of di- and trivalent cations in the aqueous subphase; the calcium ion shows an especially high effect, compared to the other ions studied. The presence of the steroid on the lipid modifies the specific area of the molecules of unsaturated lipids, which is not the case with saturated lipids, probably due to differences in the lipophilic cohesion.

Air↗
PubMed1987

Restriction enzyme accessibility and RNA polymerase localization on transcriptionally active SV40 minichromosomes isolated late in infection.

The transcriptionally active SV40 minichromosomes isolated late in infection contain a nucleosome-free ORI region or gap. To analyze the chromatin structure of this subpopulation of minichromosomes extracted at different ionic strengths in the early and late coding regions, minichromosomes were isolated in the presence of a 5, 50, or 130 mM concentration of monovalent cations and subjected to in vitro RNA elongation in either the presence or the absence of high salt and anionic detergent. The minichromosomes isolated at low ionic strength were transcriptionally more active than those isolated at physiological ionic strength. Nevertheless, in each case, the in vitro elongation complexes were present essentially on the late strand of the SV40 genome and localized preferentially in the late and 3' early coding regions. These regions were transcribed similarly in either the presence or the absence of chromatin denaturing agents. In contrast, the in vitro elongation activity of the RNA polymerase molecules present on the late strand in the middle and 5' end of the early coding region was inhibited in the absence of treatments to disrupt chromatin structure. In addition, as probed by restriction enzyme digestion, the ORI and late coding regions of the transcriptionally active minichromosomes were found to be more sensitive than the 5' region of the early genes. Taken together, these results suggest that the 5' and middle regions of the early genes of the SV40 transcriptional complexes isolated late in infection at low or physiological ionic strength are packaged in a more compact conformation than the rest of the genome.

Chromatin↗
PubMed1986

Transcriptionally active SV40 minichromosomes are restriction enzyme sensitive and contain a nucleosome-free origin region.

A nucleosome-free region or gap containing the origin of replication and the transcriptional promoter elements is observed on 20 to 25% of the SV40 minichromosomes isolated at physiological ionic strength late in infection. We used the preferential sensitivity of the gapped minichromosomes to restriction enzymes to obtain sucrose gradient fractions containing 50 to 80% of gapped molecules. The same fractions are also enriched in RNA polymerase B (II) molecules engaged in transcription. Using electron microscopy, we demonstrate here that the transcriptional complexes are preferentially sensitive to restriction enzyme digestion, which indicate that they represent a subpopulation of the gapped minichromosomes.

Chromosome Mapping↗
PubMed1986

Computer management of oligonucleotide synthesis on cellulose filters.

A computer program, OLIGO, is described which was designed to manage projects involving the synthesis of large numbers of oligonucleotide fragments using cellulose discs as immobilizing support. The program performs the functions of data entry, verification, analysis and modification; the sorting of filters before each step and project status updating after each reaction step; output of the project status at any point and storage of the finalized project data in a permanent reference file.

Base Sequence↗
PubMed1986

Cryo-electron microscopy of vitrified SV40 minichromosomes: the liquid drop model.

The structure of SV40 minichromosomes has been studied by cryo-electron microscopy of vitrified thin layers of solution. In high-salt buffer (130 mM NaCl), freshly prepared minichromosomes are condensed into globules 30 nm or more in diameter. On the micrograph, they appear to be formed by the close packing of 10 nm granules which give rise to a 10 nm reflection in the optical diffractogram. The globules can adopt many different conformations. At high concentration, they fuse into a homogeneous 'sea' of closely packed 10 nm granules. In low-salt buffer (less than 10 mM NaCl), the globules open, first into 10 nm filaments, and then into nucleosome-strings. The 'liquid drop' model is proposed to explain the condensed structure of the minichromosome in high-salt buffer: nucleosomes stack specifically on top of one another, thus forming the 10 nm filaments. 10 nm filaments in turn, tend to aggregate laterally. Optimizing both these interactions results in the condensation of 10 nm filaments or portions thereof into a structure similar to that of a liquid. Some implications of this model for the structure of cellular chromatin are discussed.

Animals↗
PubMed1986

Characterization of SV40 chromatin by mass determination on STEM.

Direct mass determination of purified SV40 minichromosomes was obtained by scanning transmission electron microscopy. Twenty to thirty percent of the minichromosomes were found with an Mr of 6.9 +/- 0.4 X 10(6). The rest of the molecules formed a spread Mr distribution ranging from 7.3 X 10(6) to 9.5 X 10(6) due possibly to different contents of the virus-coded proteins, mainly VP1. The apparent mass histogram of individual SV40 nucleosomes presents three maxima at Mr 2.1 X 10(5), 2.6 X 10(5) and 3.1 X 10(5) that could correspond to partially unravelled nucleosomes, complete nucleosomes and complete nucleosomes with the addition of VP1. Beaded structures with a higher mass were also measured; some were found at either side of the open nucleosome-free region.

Chromatin↗
PubMed1985

Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy, and subsequent TEM and STEM electron microscopy.

The three-dimensional arrangement of the chromatin components within the nucleolar organizer regions (NORs) from living oocyte nuclei was investigated. As a suitable cell system we chose vitellogenic oocytes of the orthopteran insect Acheta. This cell type is particularly attractive for analysis of nucleolar chromatin, since structural and functional aspects of NORs during early oogenic stages (including the association of NORs with amplified rDNA copies) are particularly well known (Lima-de-Faria 1974). In the course of the present study we first identified putative chromosomal NORs in isolated nuclei of mid-diplotene oocytes according to morphological characteristics using differential interference contrast (DIC) or phase contrast light microscopy. The presence of actively transcribing chromosomal NORs during this late stage of Acheta oogenesis obviously had been overlooked by previous investigators, probably due to difficulties of chromosome visualization. For a more detailed ultrastructural analysis, NORs were gently sedimented from opened nuclei and processed for sectioning using a modified "end-embedding" procedure (Mott and Callan 1975; Spring and Franke 1981). A small number of thick and thin sections could be made from individual NORs. Sections were analyzed by light microscopy, conventional transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). Whereas the structural connection of NORs to the chromosome axis and also the general arrangement of active nucleolar genes within the NOR complex could be seen with TEM, the visualization of individual nucleolar genes and the organization of transcription complexes was only possible using bright field STEM of thick sections at low temperature.

Animals↗