Artifacts observed in critical-point-dried preparations of human chromosomes by electron microscopy.
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Biomedical subjects
Publications and source records attributed to G F Bahr.
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The dry mass of individual vaccinia virions, as an example of a presumably uniform biological population, prepared in different lost at the Institute for Tropical Hygiene in Hamburg, was tested for variability by quantitative electron microscopy. A value of 5.26 X 10(-15) g for the median weight of the particle was calculated from 7,300 determinations. By assessing the variability of polystyrene latex spheres, which were used as mass standards, we demonstrated that the variability of dry mass of vaccinia virions is fivefold greater than the variability (standard) introduced by the method for determining mass. It was concluded that while genetic control in a presumably homozygous virion is strict with respect to quality, quantity of viral components (other than DNA) varies in fashion that can be aptly described by a log-normal distribution. It is recognized that this observation is empirically supported by the paradigm that any composite biological entity is subject to quantitative variability, the more so the heavier the individual representatives of a species are. In addition, the effects that extractions and staining have on the dry mass of vaccinia are reported, as well as is the median for the dry mass of other strains of pox viruses.
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As a pilot experiment towards the reconstruction of human chromosomes from their electron microscopic projections, a chromosome model was photographed and several cross-sectional planes successfully reconstructed. Some practical constraints and conditions for this type of work are defined.
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Pairs of centrioles are a frequent finding in whole-mounted, critical-point-dried chromosome preparations from normal and irradiated human lymphocyte cultures. Most frequently they are found in association with a group C chromosome, although apparently free diplosomes are not uncommon. Fibers that in every respect are the morphologic equivalent of those seen in the body of the chromatid connect to the thick-walled open-ended part of the parent centriole. These features argue for the possibility that fibers connecting centrioles are an integral part of nuclear or chromosomal chromatin. It was observed that the smaller (daughter) centriole was connected and held in the well-known angular configuration to the thick-walled opne end of the parent centriole by a few, probably only two, colchine-resistant fibers.
Electron microscopy, with sodium phosphotungstate as negative stain, has been carried out on purified jackbean urease prepared at three levels of quaternary structure: (a) A1 urease, Mr = 240 000, S20,W = 11.5 S (b) alpha urease, Mr = 480 000, S20,w = 18.3 S (c) polymers of alpha urease above the tetramer stage. The compatibility of the images from level to level leaves no doubt that the enzyme itself is being visualized, and the following geometry is suggested by electron microscopy: A1 molecules are cyclic trimers, which pair up in eclipsed position across a 1-nm cleft to form the hexameric alpha, which displays D3 (or 32) symmetry of a trigonal prism. Polymers consist of alpha molecules aligned with their clefts coplanar and an angle of 120 degrees between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which have indicated a hemispherical A1, a spherical alpha, and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were found to be compatible with the rosette, straight-chain, and zig-zag forms seen in the electron microscope, and with the suggested protomer arrangement in A1 and alpha urease.
The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles.
When the chromosomes of the Indian muntjac, Muntiacus muntjak, were compared following treatment with two presently used banding methods, trypsin-Giemsa (G) and quinacrine-hydrochloride (Q) with structural bands as seen in the electron microscope, definite correlations were observed with respect to the numbers and positions of individual bands. - Weights obtained for the individual chromosomes were: No. 1, 9.98 pg; No. 2, 4.10 pg; No. 3, 4.43 pg; No. 3-X, 5.05 pg; and Y, 0.55 pg. Average diameters and weights for individual fibers were 193 A and 8.74 times 10-16 g/micron, respectively, for stimulated metaphase chromosomes and 185 A and 8.73 times 10-16 g/micron, respectively, for unstimulated chromosomes. Fibers of interphase nuclei exhibited an average diameter of 191 A and a weight of 5.87 times 10-16 g/micron. - The total amound of nuclear DNA present in interphase nuclei was 3.88 pg.
Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was localized to chromatin fibers of Raji cells. When EBNA was labeled with the horseradish-peroxidase technique of Sternberger a strong reaction covered most fiber surfaces as observed by electron microscopy. The dry mass of Raji chromatin increased after exposure to both EBV-positive and -negative serum, but the increase was significantly larger in the material treated with positive serum.
Human chromosomes as a type-sample for mammalian chromosomes consist of 200-A fibers, folded to chromomeres, which are interconnected by about a dozen longitudinal fibers. The average fiber at both interphase and metaphase contains 28.3 lengths of one double helix of DNA per length of fiber. The orientation of DNA imparts polarity to the fiber and thus to the chromosome and is an important constraint in concepts of chromosomal aberrations and rearrangements, some of which are being interpreted on the basis of fiber-fiber exchanges. Chromosomal rearrangements discernible by light microscopy are not likely to be fully synonymous with change in gene sequence. Chromosomes are considered to possess a plane of symmetry originating from semiconservative replication. Implications for chromosomal structure, centromeric function, and chromatid cohesion are discussed. Fibers connecting one chromosome to others are discussed in light of the proposal that fiber regions of repeated nucleotide sequences exist that facilitate fiber-fiber exchanges. No free fiber or DNA ends are thought to occur at any time in the nucleus.
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