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Employing stereo-electron microscopy on sections (ca. 0.1 micrometer thick) of the ciliated protozoa Stylonychia, Oxytricha and Euplotes, we have examined the ultrastructure of macronuclear replication bands (RB) and micronuclei. The macronuclear RB is composed of two zones: a "forward zone" showing a special chromatin organization consisting of regular 40 to 50 nm diameter fibers with an indication of nucleosomal substructure; and a "rear zone", the site of DNA replication, consisting of a mesh of 10 nm chromatin fibers. Micronuclei exhibit chromatin strands of 60 to 70 nm diameter. Employing the Bernhard staining procedure, we have observed that the chromatin fibers of the RB and of the micronuclei remain unbleached, whereas macronuclear condensed chromatin is bleached of stain, indicating that the replication band and the micronuclei contain chromatin in a configuration different from that of other forms of densely packed chromatin. The regularity of the chromatin fibers within the forward zone is of particular interest since it is comparable to the regularity of chromatin seen in the transcriptionally-inactive chromatin of other nuclei such as avain erythrocytes and sea urchin spermatozoa. It is likely that the forward zone chromatin fibers consist of highly ordered arrangements of nucleosomes, associated with additional nonhistone proteins.

A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.

Stereo electron microscopy was employed to examine thin sections of Chinese hamster ovary metaphase chromosomes in situ and of chromosomes released from Chinese hamster ovary cells by several methods. Detergent lysis of cells in a buffer containing Mg++ and Ca++ and hypotonic lysis of cells in a hexylene glycol-Ca++ buffer released chromosomes that exhibited a three-dimensional meshwork of about 50 nm chromatin fibers. Fragmentation of cells in serum-free medium by vortexing with glass beads revealed a more dispersed chromosomal morphology with a mesh of 10 to 25-nm fibers exhibiting a presumptive nucleosomal substructure. Possible origins of the various fiber sizes are discussed in terms of current models of metaphase chromosome structure.

Thick sections (0.5-1.0 micron) containing pilocarpine-hyperstimulated Balbiani ring (BR) genes of Chironomus tentans salivary glands were reconstructed by electron microscope tomography (EMT). Six transcription loops were analyzed at low resolution permitting a measurement of transcription unit length in situ. Comparison of the average transcription unit length with the BR gene coding region DNA length yields an estimate of the DNA compaction ratio in situ. The estimated DNA compaction ratio (approximately 3-fold) is consistent with a significant degree of nucleosome unfolding in the hyperstimulated BR genes.

Chironomus tentans fourth instar larvae were awakened from oligopause (diapause) and treated with pilocarpine to stimulate Balbiani ring transcriptional activity. Salivary glands were fixed, sectioned and examined by conventional and stereo-electron microscopy. Balbiani ring RNP lateral fiber densities were measured in well-formed regions with clear Balbiani ring granules arranged along chromatin transcription axes. Despite an up to ten-fold range in Balbiani ring transcriptional activity [10, 12, 14], we observed no significant difference in lateral fiber densities. These data are discussed in terms of the variety of mechanisms for modulating overall transcriptional activity, including recruitment of parallel transcription units in the polytene chromosome. The lack of other major ultrastructural changes at the nuclear envelope and within the domains of tubular rough endoplasmic reticulum argue strongly against a major dismantling of subcellular structure during the induced oligopause.

Cross-sectional views of mature regions of Balbiani Ring (BR) transcription loops were reconstructed by electron microscope tomography. Balsa wood models were built based upon the resulting tomograms. Coordinates of the centers of the ribonucleoprotein BR granules were estimated and employed to calculate the approximate orientation of the BR transcription unit axis in the plastic section. The density of BR granules per micron of transcription unit axis was estimated. Employing structural parameters for mature regions of BR transcription loops derived from present and previous studies, theoretical calculations were performed to examine potential steric restrictions around the central chromatin axis. Tomograms were also observed and photographed on a varifocal (vibrating) mirror to illustrate the utility of a rapid user-interactive 3-D display.

Employing stereo-electron microscopy on sections (ca. 0.1 micrometer thick) of isolated salivary glands from Chironomus tentans, we have formulated structural models for the organization of the peripheral nucleolar strands, the Balbiani ring (BR) transcription axes, and the tubular rough endoplasmic reticulum (RER). (i) The ca. 67 nm wide peripheral nucleolar strands display a higher-order organization of ca. 13.6 nm granules connected to a central axis, possibly representing a storage of pre-ribosomal RNP. (ii) The BR transcription axes were visualized in three-dimensions, and the density of lateral RNP fibrils was estimated (ca. 36 RNP fibrils/micrometer transcription axis). This estimate of the density of nascent RNP fibrils, suggests that in situ the DNA packing ratio of transcribing regions in intermediate between extended DNA and a ca. 10 nm polynucleosomal fiber. (iii) The tubular RER appears to reveal extended helical arrays of polyribosomes. The helices have an overall diameter of ca. 130 nm and exhibit a mean of 12 ribosomes per helical turn. We suggest that this arrangement of membrane-bound ribosomes represents a functional specialization of the cell, facilitating the translation of the very large BR mRNA of ca. 37 kb into a secretory product.

Utilizing the techniques of stereo-electron microscopy (stereo-EM) and energy-dispersive x-ray analysis (EDX), we have studied aspects of the ultrastructure of avian reticulocytes. Stereo-EM of thin sections (0.10 to 0.25 mum thick) stained with uranyl and lead revealed the three-dimensional arrangement of 25 nm chromatin fibers on the tangential surfaces of nuclei. Use of the Bernhard staining procedure in combination with stereo-EM permitted a three-dimensional view of the interchromatin spaces and channels leading to the nuclear pores, and of cytoplasmic polyribosomes. With either staining technique we frequently encountered in the cytoplasm clusters and paracrystalline arrays of electron-dense granules with granule diameters approximately 1/2 that of monomer ribosomes. These granules were highly electron-dense in unstained specimens and have been identified as intracellular ferritin on the basis of the similarity of their ultrastructural morphology to that of horse spleen ferritin and their high content of iron as determined by EDX. The possibility that these granules represent toxic products of phenylhydrazine treatment (Heinz bodies) is considered unlikely, since we have demonstrated in this study that Heinz bodies cannot be visualized in unstained preparations, do not reveal the same granular structure, and do not contain significant amounts of iron above background. The occurrence of intracellular ferritin is discussed in light of current concepts of iron transport and storage during erythropoiesis.