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Biomedical subjects

J Dubochet

Publications and source records attributed to J Dubochet.

At least 19 recordsLinked to original sources
PubMed1992

The apical localization of transcribing RNA polymerases on supercoiled DNA prevents their rotation around the template.

The interaction of Escherichia coli RNA polymerase with supercoiled DNA was visualized by cryo-electron microscopy of vitrified samples and by classical electron microscopy methods. We observed that when E. coli RNA polymerase binds to a promoter on supercoiled DNA, this promoter becomes located at an apical loop of the interwound DNA molecule. During transcription RNA polymerase shifts the apical loop along the DNA, always remaining at the top of the moving loop. This relationship between RNA polymerase and the supercoiled template precludes circling of the RNA polymerase around the DNA and prevents the growing RNA transcript from becoming entangled with the template DNA.

Binding Sites↗
PubMed1991

Spatial visualization of DNA in solution.

An image analysis method is presented which allows for the reconstruction of the three-dimensional path of filamentous objects from two of their projections. Starting with stereo pairs, this method is used to trace the trajectory of DNA molecules embedded in vitreous ice and leads to a faithful representation of their three-dimensional shape in solution. This computer-aided reconstruction is superior to the subjective three-dimensional impression generated by observation of stereo pairs of micrographs because it enables one to look at the reconstructed molecules from any chosen direction and distance and allows quantitative analysis such as determination of distances, curvature, persistence length, and writhe of DNA molecules in solution.

DNA, Bacterial↗
PubMed1991

A model for cryosectioning based on the morphology of vitrified ultrathin sections.

Electron microscopy of vitrified ultrathin sections allows cell ultrastructure to be studied in the hydrated state. Sectioning of the frozen material is, however, a limiting step, since the cutting forces cause severe mechanical deformation. In order to address this problem, we have investigated the surface of cryosections. It is shown that cryosections have two fundamentally different surfaces. One surface is rough, deformed by cutting-induced deformation lines which are orientated perpendicular to the cutting direction. The other surface, in comparison, is not affected by those deformation lines. Except for knife marks it is smooth. In order to explain the observations, the following model is proposed. The rough relief corresponds to the former block face. Its roughness originates from material that is squeezed out of the section plane when the section is compressed in the cutting direction and bent away from the specimen block. The smooth section surface is the surface in contact with the knife during the sectioning. This contact keeps the surface smooth while imprinting the knife marks.

Animals↗
PubMed1991

Freezing: facts and hypothesis.

Hexagonal ice crystals formed in frozen biological specimens are large and branched. They can produce severe structural damage by solute segregation but there are also cases where they seem to cause only minor damage. When cooling is more rapid, cubic ice crystals can be formed. These are small and in general, they cause little damage. These observations can be readily explained with the hypothesis that large hexagonal ice crystals can originate from the rewarming induced transformation of a large number of cubic ice crystals. This transformation would take place without significant solute displacement.

Animals↗
PubMed1990

Direct visualization of supercoiled DNA molecules in solution.

The shape of supercoiled DNA molecules in solution is directly visualized by cryo-electron microscopy of vitrified samples. We observe that: (i) supercoiled DNA molecules in solution adopt an interwound rather than a toroidal form, (ii) the diameter of the interwound superhelix changes from about 12 nm to 4 nm upon addition of magnesium salt to the solution and (iii) the partition of the linking deficit between twist and writhe can be quantitatively determined for individual molecules.

DNA, Superhelical↗
PubMed1990

Evaporation during preparation of unsupported thin vitrified aqueous layers for cryo-electron microscopy.

Evaporation of water cannot be fully avoided when an unsupported thin vitrified film of an aqueous suspension is prepared for cryo-electron microscopy. This results in increasing concentration of solute which could affect the observed material. We have quantitatively studied this effect by measuring the contrast of polystyrene spheres in a metrizamide solution. The drying effect is generally negligible when specimens are prepared on a hydrophilic perforated support but it is frequently important when hydrophobic films are used instead. A flow of humid air, double blotting with minimal exposure of the thin liquid film to the atmosphere, or an automatic plunger optimizing the blotting conditions are simple methods for reducing drying effects. With this third device acting on a hydrophilic supporting film, the increase of solute concentration is limited to less than 20%.

Cold Temperature↗
PubMed1989

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.

Actins↗
PubMed1988

Head structure of bacteriophages T2 and T4.

The length-to-width ratios of bacteriophage T2 and T4 heads and stereometric angles specifying the prolate icosahedral T2 capsid were evaluated on electron micrographs recorded from samples prepared by a variety of methods. The copy numbers of the major capsid protein, gp23*, of T2 and T4 phages were compared by quantitative gel electrophoresis. Taken together, the resulting values are most compatible with triangulation numbers T = 13 and Q = 21 for both T2 and T4, thus confirming the previously proposed capsid architecture of T4 revealed by indirect measurements and thereby eliminating the repeatedly reported discrepancy between T2 and T4 in favor of a common Q number of 21 corresponding to 960 copies of gp23*.

Capsid↗
PubMed1988

Length and shape variants of the bacteriophage T4 head: mutations in the scaffolding core genes 68 and 22.

The shape and size of the bacteriophage T4 head are dependent on genes that determine the scaffolding core and the shell of the prohead. Mutants of the shell proteins affect mainly the head length. Two recently identified genes (genes 67 and 68) and one already known gene (gene 22), whose products are scaffold constituents, have been investigated. Different types of mutants were shown to strongly influence the proportion of aberrantly shaped particles. By model building, these shape variants could be represented as polyhedral bodies derived from icosahedra, through outgrowths along different polyhedral axes. The normal, prolate particle is obtained by elongation along a fivefold axis. The mutations of the three core genes (genes 67, 68, and 22) affect the width mainly by lateral outgrowths of the prolate particle, although small and large isometric particles are also found. Many of the aberrant particles are multitailed, suggesting a correlation between tail attachment sites and shape.

DNA Mutational Analysis↗
PubMed1987

Organization of double-stranded DNA in bacteriophages: a study by cryo-electron microscopy of vitrified samples.

In this paper it is shown that conformation and packing of double-stranded DNA within the head of bacteriophages lambda and T4 can be assessed by cryo-electron microscopy of vitrified specimens. Electron diffraction patterns show that DNA within vitrified bacteriophages has a B conformation. Electron micrographs of vitrified bacteriophages show domains within the head formed by a approximately 2.5-nm striation and arising from the DNA packing. The number of differently oriented domains seen within a vitrified bacteriophage depends upon the geometry of the DNA container: the bacteriophage capsid. The packing of DNA within bacteriophages seems then to be governed by at least two phenomena. The first is the tendency of DNA to form local alignments (nematic liquid crystals). The second is the orientation of these liquid crystals by the bacteriophage capsid. From these observations we propose a possible packaging mechanism: constrained nematic crystallization.

Bacteriophage lambda↗
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

Cryo-electron microscopy of vitrified chromosomes in situ.

Chromosomes of metaphase-arrested Chinese hamster ovary (CHO) and HeLa cells were examined in situ, unfixed and unstained, by cryo-electron microscopy. In hydrated, vitrified cryo-sections, chromosomes exhibit a characteristic homogeneous, grainy texture, which, on optical diffraction, gives rise to a broad reflection corresponding to 11 nm. No superstructure or periodic order is discernible. These observations suggest that the chromosome is formed by the compact association of 11 nm filaments, or portions thereof, interacting in a manner akin to the molecules of a liquid. Some implications of the liquid model of chromosome structure are discussed.

Animals↗