Protoplast formation and cell wall regeneration in Clostridium perfringens.

A protocol was developed for the efficient production and regeneration of Clostridium perfringens protoplasts. Cell wall regeneration frequencies of up to 5% were obtained.     

Electron microscopic studies of aggregation and pellicle formation inAzospirillum spp.

Microscopic studies on aggregation ofAzospirillum brasilense Cd and the local isolate Cd-1 revealed the presence of an extracellular layer (ECL) on the outer surface of cells taken from the early exponential phase. Incubation of the bacteria with cationized ferritin (CF) was followed by labelling of the ECL of both Cd and Cd-1 taken from the stationary phase. Concanavalin-A-ferritin (Con-A-F), a lectin conjugated with ferritin, was bound to the ECL which was developed during the stationary phase, but not the exponential phase, in both isolates. In static liquid cultures,Azospirillum brasilense Cd formed lateral flagella and fimbriae-like structures which were not observed in shaking liquid cultures. It is suggested that these filaments may play a role in cell-to-cell adhesion of Azospirillum that results in pellicle formation observed in static cultures.     

Electron microscopic identification of the yeast spliceosome.

We have partially purified the yeast spliceosome by differential sedimentation in glycerol gradients. By electron microscopy we have identified a particle in these fractions that is the spliceosome. In 100 mM KCl buffer, the yeast spliceosome is an ovoid disc with the dimensions of 20 x 23.5 nm with a central indentation. To verify that these ovoid particles were spliceosomes, specific labels were used to tag them. These tagged spliceosomes were then identified in the electron microscope. The salt dependent shift of sedimentation rate for the spliceosome can be explained by a change in size of the particle.     

Protoplast formation and cell wall regeneration in Clostridium perfringens

A protocol was developed for the efficient production and regeneration of Clostridium perfringens protoplasts. Cell wall regeneration frequencies of up to 5% were obtained.     

Protoplast formation and cell wall regeneration inClostridium thermocellum

Summary A protocol was developed for the production ofClostridium thermocellum protoplasts and the regeneration of protoplasts into vegetative cells. Protoplasts were prepared by exposure of whole cells to lysozyme (125 ug/ml for 10 min. at 45 C). Protoplasts plated on regeneration agar reverted to walled cells with a frequency of up to approximately 0.3 % of input cells.     

Electron microscopic studies of condensed mitotic chromosomes in the fission yeast Schizosaccharomyces pombe

By blocking cells in mitosis with the anti-fungal drug thiabendazole, it has been possible to carry out ultrastructural studies on the condensed chromosomes of the fission yeast, Schizosaccharomyces pombe. It is estimated that the DNA in these chromosomes is compacted approximately 1000-fold, and that the nucleoprotein density is similar to that of higher eukaryotic metaphase chromosomes. A basic structural component of the condensed chromosomes appears to be a 50-60 nm fibre, which is often visible in a loop configuration on the periphery of the chromatids. This is reminiscent of the 50-60 nm fibre loops which are frequently seen in preparations of metaphase chromosomes, and suggests that mechanisms of nucleoprotein folding may be similar in both lower and higher eukaryotes.     

Electron microscopy of the cotton fibre: new observations on cell wall formation.

The ultrastructure of the cotton fibres was examined after developing successful fixation methods. Fibre cells were fixed at different stages of development. In cells which were elongating and producing primary cell walls, the Golgi apparatus appeared to be directly involved in secretion and synthesis of primary wall components. In cells which were synthesizing thick secondary cell walls, evidence suggested a major role for the endoplasmic reticulum and plasma memebrane in the synthesis and secretion of secondary wall materials. The possibility of a shift from a Golgi apparatus pathway for primary wall synthesis to an endoplasmic reticulum pathway for secondary wall synthesis is discussed. Plasma membrane micro-invaginations are present only during secondary wall synthesis and may represent sites of cellulose assembly. A model for primary wall biogenesis via the Golgi apparatus is presented, and the potential of the cotton fibre as a model system for studying cellulose biogenesis in higher plants is discussed.     

Electron microscopic observations of cell regeneration from cultured protoplasts ofAmmi visnaga

Summary Protoplasts ofAmmi visnaga initiated cell wall formation within 2 days in culture; after 13 days the new cells were enclosed by a cell wall similar to the walls on the original cultured cells. Budding occurred in protoplasts with little or no detectable cell wall. No evidence was obtained for direct participation of any organelle in cell wall formation. The cytoplasm of regenerating cells contained numerous organelles and appeared typical of actively growing plant cells; they were easily distinguished from degenerate cells and protoplasts. While coated vesicles were common, spiny vesicles occurred in only a few cells. Sustained cell division yielded multicellular aggregates. Multinucleate protoplasts, formed by spontaneous fusion, did not divide; some of them contained annulate lamellae with few pore complexes.Supported by the National Research Council of Canada, Grant A6304.     

Electron microscopic studies on radiation-induced changes of cell surface negative charges

The amount and distribution of negatively charged sites of different cells (human fibroblasts, B16 melanoma cells, a human lymphoid leukemic cell type) were investigated. In the non-irradiated fibroblasts and B16 melanoma cells the negatively charged sites were localized mainly on apical and lateral surfaces as well as in a polarized manner. However, negatively charged sites on the control human lymphoid leukemic cells often have patched distribution. It was demonstrated that the amount and distribution of negatively charged sites on primary human fibroblasts and B16 melanoma cells changed upon ionizing radiation. However, the topology of negative charges on investigated human leukemic cell membrane did not change.     

Integrative studies put cell wall synthesis on the yeast functional map

The fungal cell wall field, traditionally focused on polysaccharide composition and synthesis, retains a certain static architectural imagery of structural rigidity and integrity, with the wall offering protection from a harsh environment. This picture of the wall is increasingly changing to that of a bustling construction site, as research uncovers the organizational complexity of its assembly. With recent molecular and genomic studies on Saccharomyces cerevisiae, cell wall synthesis and biology appear increasingly to be dynamic and adaptable processes that are fully integrated with the underlying cytoskeletal and polarity machinery that drive cell cycle progression.     

Electron microscopical studies of frozen-dried yeast: II. The nature of basophile particles and vesicular nuclei in Saccharomyces

Haploid and diploid vegetative nuclei of Saccharomyces were stained selectively for basophilia after post-fixation in vacuo with anhydrous, vapor reagents specifically effective for proteins and polypeptides and studied in conjunction with enzymatic and other controls. The nuclei cleave simply by elongation and medial constriction of hour-glass forms with no indications of a spindle apparatus or resolvable chromosomes with classical morphological attributes, or a breakdown of the nuclear boundary at any time until final cleavage. Instead, the nuclear contents are particulate, measuring about 50–80 Å and seemingly dispersed at random in most sectioning planes, but frequently detectable as short rows streaming into the developing daughter nucleus. Association of these particles with faintly stainable submicroscopic cords of anastomotic form in sections is detectable; one or two peripheral clusters of similar particles, of hitherto undetermined function, occur in the dividing nucleus regardless of haploidy or diploidy. No organization of obviously light microscopically resolvable dimension occurs in the nucleus. Integrity of the particulate matter in the nucleus is destroyed or very greatly reduced by DNA-ase, but preserved after RNA-ase. The latter abolishes basophily of the dense deposits of ribosomes in the cytoplasm with the exception of a small resistant fraction mostly in the vicinity of the nucleus. These resistant particles are not seen in specimens subjected successively to both the nucleic acid depolymerases. The cytogenetical implications of these findings are discussed very briefly in view of Mendelian heredity established in yeast, and in terms of an evolution of cytogenetical mechanisms.     

Electron microscopic observations of surface projections on Chlamydia psittaci reticulate bodies.

Electron microscopic observations were carried out to confirm the presence of surface projections on Chlamydia psittaci reticulate bodies (RBs). The morphology of the projections on RBs was identical with that on elementary bodies (EBs); one end of each projection was connected with the cytoplasmic membrane, but the other end of the projection protruded beyond the cell wall through a fine hole or rosette in the cell wall. The results demonstrated that the rosettes seen in RB cell walls were morphological markers indicating the presence of the surface projections. A statistical anaylsis of the number of projections on EBs and the number of rosettes in RB cell walls prepared at 10, 15, and 20 h after infection demonstrated that all RBs had the projections and that the number of projections was maximal by 10 h after infection and then decreased gradually to approximately the same number of projections on EBs.