3D analysis of chromosome architecture: advantages and limitations with SEM.

Three-dimensional mitotic plant chromosome architecture can be investigated with the highest resolution with scanning electron microscopy compared to other microscopic techniques at present. Specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis. Applied to investigate the structural effects of different preparative procedures, these techniques were the groundwork for the "dynamic matrix model" for chromosome condensation, which postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.

Electron diffraction studies of the peptidoglycan of bacterial cell walls.

Peptidoglycan of the gram-positive bacterium Lactobacillus plantarum was adsorbed onto hydrophilic crystalline films of graphitic oxide. Destruction by radiation damage was reduced by using a device which permitted scanning of the specimen for several hours through a focussed electron beam of low current density (2.5 X 10(-5) A/cm2). In additition to the sharp Debye-Scherrer rings caused by graphitic oxide, peptidoglycan causes a diffuse Debye-Scherrer ring in the region of 4.5 A. This can be interpreted as the packing periodicity of the peptidoglycan within the planes of the peptidoglycan sacculi.

Surface layers of Xanthomonas malvacearum, the cause of bacterial blight of cotton.

Mureins were isolated from two strains of Xanthomonas malvacearum, a phytopathogenic bacterium causing bacterial blight of cotton. The purity of murein was 70-95 % and the amino acid and amino sugar components (glutamic acid, alanina, meso-disminopimelic acid, muramic acid and glucosamine) were present at the molar ratio of 1:1.9:1:l.12.0.85. The bacterium secreted a copious amount of slime which masked itd surface structure. The slime was composed of densley interwoven network of filamentous material originating from the cell surface and extended into the medium without and discernable boundary. The slime was secreted through surface layers pores by force, giving the effect of a spray or jet. Slime also played a role in chain formatin of baterial cells.

Possible orientation of the fatty acid chains in lipopolysaccharide.

The fatty acid chains of the lipid A component of lipopolysaccharides are hexagonally packed with a lattice periodicity of 4.1 A. The smallest subunit of this lattice consists of a disaccharide to which seven fatty acid chains are linked representing the hydrophobic part. Carbohydrates and substituted phosphate residues linked to them form the hydrophilic part of the molecule. Because of sterical reasons and because of the necessity of a separation of hydrophobic and hydrophilic part, we could derive that the angles of the hydrocarbon chains with the planes of the two sugar residues should be as vertical as possible and the planes of both sugar residues should be approximately coplanar forming an angle of about 180 degrees with one another. Conformation angles for all the theoretically possible linkages of the disacoharide and of the linkages of the fatty acid residues have been calculated. Between the two-N-acylglucosamine residues theoretically beta-1,4, alpha-1,6 or beta-1,6 linkages are possible. The experimentally found beta-1,6 linkage has the largest degrees of freedom for its conformation angles.

A three dimensional model of the digestion of peptidoglycan by lysozyme.

The digestion of single peptidoglycan chains of the recently proposed conformation (Formanek et al., 1974) can be described with the same enzymatic mechanism as proposed by Phillips for a hexasaccharide consisting of alternating N-acetylglucosamine, N-acetylmuramic acid residues (Phillips, 1966). It is shown by model building, that in a peptidoglycan lysozyme complex the peptide chains do not exhibit any sterical hindrance. The digestion of the peptidoglycan sacculus by lysozyme may occur at latice defects of its paracrystalline structure. A slit of about 30 A length and 10--15 A width between peptidoglycan micells may be sufficient for the attachment of lysozyme.

A new chromosome model.

We present a new model of the three-dimensional structure of chromosomes. With DNA and protein staining it could be shown by high-resolution scanning electron microscopy that metaphase chromosomes are mainly composed of DNA packed in "chromomeres" (coiled solenoides) and a dynamic matrix formed of parallel protein fibers. In the centromeric region, the chromomeres are less densely packed, giving insight into the matrix fibers. We postulate that chromosome condensation is achieved by the binding of solenoids to matrix fibers which have contact sites to one another and move antiparallel to each other. As condensation progresses, loops of solenoids accumulate to form additional chromomeres, causing chromosomes to become successively shorter and thicker as more chromomeres are formed. For sterical reasons, a tension vertical to the axial direction forces the chromatids apart. The model can simply explain the enormous variety of chromosome morphology in plant and animal systems by varying only a few cytological parameters. Primary and secondary constrictions and deletions are defined as regions devoid of chromomeres. Even in the highly condensed metaphase, all genes would be easily accessible.

Imaging of DNA in human and plant chromosomes by high-resolution scanning electron microscopy.

We describe a DNA-specific staining procedure, using a blue platinum organic dye, which allows DNA imaging of chromosomes by detection of back-scattered electrons in the scanning electron microscope. DNA distribution is visualized within chromosomal details of the centromeric and satellite regions, or in interphase chromatin, with high-resolution (10-15 nm) for comparison with the corresponding secondary electron image representing DNA plus protein.

Signal function of metabolism of neutral amino acids and 2-keto acids for initiation of insulin secretion.

2-Ketocaproate and 2-ketoisocaproate were equally potent insulin secretagogues. The insulin secretory potency of L-leucine was less than half of that of the keto acids and L-norleucine did not induce any insulin release by isolated islets and by the perfused pancreas from ob/ob mice. Rates of decarboxylation of 2-keto-[1-14C]isocaproate and of 2-keto-[1-14C]caproate were equally high. The finding is consistent with the view that enhanced production of reducing equivalents is necessary for initiation of insulin release. The rates of decarboxylation and transamination of L-[1-14C]leucine by isolated pancreatic islets were several times higher than the rates observed with L-[1-14C]norleucine. Thus, the high activity of the pancreatic islet branched-chain amino acid aminotransferase may be important for the recognition of L-leucine as an insulin secretagogue by pancreatic B-cells.

Localization of aggregation substances of Enterococcus faecalis after induction by sex pheromones. An ultrastructural comparison using immuno labelling, transmission and high resolution scanning electron microscopic techniques.

The distribution of sex pheromone induced aggregation substance was studied on the cell surface of various Enterococcus faecalis strains. In the accompanying paper we have shown that the aggregation substance appears as a layer of hairlike structures. Using direct and indirect immunogold technique, transmission electron microscopy and high resolution scanning electron microscopy we investigated the appearance and distribution of the aggregation substance. The "hairs" increase in number with increasing exposure to sex pheromones (maximum density: 1300/microns2). We show that these structures are unequally distributed over the cell surface, even if the cells were induced by sex pheromones for a long period of time. Statistical analysis of the unequal distribution indicates that aggregation substance is incorporated into pre-existing "old" cell-walls and that this incorporation shows a saturation ca. 40 min after addition of sex pheromones.

[Structural investigations on lipopolysaccharides of mutants S SF 1111 and R 595 SF 1167 of Salmonella minnesota (author's transl)]. / Strukturuntersuchung mit Röntgenbeugungsmethoden an Lipopolysacchariden von Salmonella minnesota Mutanten S SF 1111 und R 595 SF 1167.

The lipolysaccharides of two mutants of Salmonella minnesota with known chemical structure were investigated by X-ray methods. Both thickness of the lipopolysaccharide layer and arrangement of the molecular components could be estimated. Using these informations a space-filling model of the structure was built.

The ontogeny of lipid bodies (spherosomes) in plant cells : Ultrastructural evidence.

Maturing embryos of 16 oil plants, anise suspension culture cells, and Neurospora crassa cells were prepared for electron microscopy at different stages during massive lipid accumulation. Lipid-rich structures of certain species were best preserved by dehydration of fixed tissues in ethanol without propylene oxide, embedding in Spurr's Medium, and polymerization at room temperature. In all cells examined, spherical lipid bodies (spherosomes) showed a moderately osmiophilic, amorphous matrix and displayed a delimiting half-unit membrane when sectioned medially. Associations with the endoplasmic reticulum (ER) were viewed at any stage during lipid body development but with different frequency in the different plant species. Plastids of fat-storing cells exhibited conspicuously undulate outer and inner envelope membranes that formed multiple contact sites with each other and protuberances into both cytoplasm and stroma. Some species, e.g., Linum, have plastids with tubular structures that connect the inner membrane to the thylakoid system; in addition, in the stroma vesicles fusing with or apparently passing through the envelope were observed. The outer envelope membrane may be associated with ER-like cytoplasmic membrane structures. In addition, lipid bodies of various sizes were found in contact with the plastid envelope. The ultrastructural observations are interpreted to match the published biochemical evidence, indicating that both plastids and ER may be involved in the synthesis of storage lipids and lipid body production.

Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy.

The surface structure of mitotic barley chromatin was studied by field-emission scanning electron microscopy (FESEM) and scanning force microscopy (SFM). Different stages of the cell cycle were accessible after a cell suspension was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated specimen (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of the residue chromatin during mitosis was within the range of 65-85 microm(3). A comparison with the theoretically calculated volume indicated a contribution of about 40% of internal cavities. Decondensation of chromosomes by proteinase K led to a drastic decrease in the chromosome volume, and a 3-D netlike architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM without any intermediate drying step. A granular surface was obvious but with an appreciably lower resolution. Under similar imaging conditions proteinase K-treated chromosomes exhibited low topographic contrast but were susceptible to plastic deformations.