Disordered mitoses and extranuclear chromatin in the cells of normally developing embryos of Brachydanio rerio

In the cells of normal embryos of zebrafish, Brachydanio rerio, the Feulgen-positive particles were found in the cytoplasm, in addition to the following abnormalities of mitoses: retardation and loss of chromosomes, disorganization of mitotic figures similar to those seen with C-mitosis, condensation and pycnotic transformation of chromatin preceding the cellular death. The dynamics of these phenomena was traced in the course of development starting from the 8 cell stage to the late blastula stage. The high frequency of the above findings, as well as a certain extent of the topological correspondence of abnormal mitoses enable the author to propose that the normal development of bony fishes is accompanied by degradation of the hereditary material in some cells of the embryo. This may constitute a step of differentiation for at least provisional structures of the embryos.     

Fine structural localization of phosphatases in cilia and basal bodies of Tetrahymena pyriformis.

Cytochemical localization of ATPase activities in cilia and basal bodies of Tetrahymena pyriformis revealed a number of possible sites of ATPases. In basal bodies, reaction product was localized on the periphery of basal body microtubules, in the core of the B-microtubules, on the dense basal body core, and on the basal plate; some reaction product was associated with the postciliary and basal microtubules. In the cilium, reaction product was associated with the ciliary membrane, the basal granule, the periphery of the outer doublet microtubules, in the core of the B-microtubules, and on the arms and either the central microtubules or the radial spoke heads. Reaction product deposition required ATP and either Ca2+ or Mg2+ or ADP and Mg2+. When incubated in the presence of ATP and Na+, reaction product was only found at the base of the cilium in the region of the ciliary necklace. Implications of the various sites of activity are discussed with respect to possible mechanisms of ciliary motility.     

The histone content of Tetrahymena ribosomal gene-containing chromatin

The histone composition of the ribosomal gene containing chromatin (rChromatin) of the ciliated protozoan Tetrahymena pyriformis has been investigated using purified nucleolar fractions in which ribosomal DNA constitutes 65-82% of the total DNA. In isolated nucleoli, rChromatin largely retains the periodic activated structure characteristic of rChromatin in the nucleus. For all five major histone classes, the histone to DNA ratios of nucleolar chromatin are similar to those of bulk macronuclear chromatin. These results argue that the differences between activated rChromatin and inactivated chromatin are not due to a deficiency in the number of histones available to form nucleosomes on the rDNA.     

Ultrastructural features of chromatin nu bodies

Spread chromatin fibers and isolated chromatin fragments prepared from chicken erythrocyte nuclei were stained with dilute aqueous uranyl acetate. High-resolution electron micrographs reveal two new morphological features exhibited by many of the chromatin nu bodies: (a) lateral association of the nu body with the connecting strand, and (b) a centrally stained spot approximately 15 A wide, possibly corresponding to a hole or crevice within the nu body.     

Conformational states of chromatin nu bodies induced by urea.

Monomer chromatin nu bodies (nu1) from chicken erythrocyte nuclei were exposed to 0-10 M urea plus 0.2 mM EDTA (PH 7). Alterations in nu1 conformation were examined using hydrodynamic methods (i.e., S, eta, and (formula: see text)), thermal denaturation, circular dichroism, reactivity of histone thiol groups to N-ethyl maleimide, and electron microscopy. The two domains of a nu body (i.e., the DNA-rich shell and the protein-rich core) aeared to respond differently to the destabilizing effects of increasing urea; DNA conformation and stability exhibited noncooperative changes; the core protein structure revealed cooperative destabilization between 4 and 7 M urea. Companion studies on the conformation of the inner histone "heterotypic tetramer" also revealed cooperative destabilization with increasing urea concentration.     

Nucleologenesis: Composition and fate of prenucleolar bodies

A time course study was conducted on nucleologenesis after release from a mitotic block in the presence and absence of actinomycin D to determine the composition and fate of prenucleolar bodies (PNBs). Prenucleolar bodies, whether naturally occurring or induced by actinomycin D treatment, stain with silver and contain phosphoproteins B23 and C23, two of the major proteins of the interphase nucleolus as determined by double label immunofluorescence with specific antibodies. The nucleolus is formed by fusion of PNBs, which subsequently reorganize and form internal fibrillar and peripheral granular regions. Actinomycin D prevents fusion of PNBs, which are then randomly dispersed throughout the nucleus but they still contain proteins B23 and C23. These results demonstrate that the nucleolus is formed by fusion of prenucleolar structures whose biochemical composition resembles the mature nucleolus, since PNBs contain at least two of the major nucleolar proteins.     

The fate of deleted DNA produced during programmed genomic deletion events in Tetrahymena thermophila.

Thousands of DNA deletion events occur during macronuclear development in the ciliate Tetrahymena thermophila. In two deleted genomic regions, designated M and R, the eliminated sequences form circles that can be detected by PCR. However, the circles are not normal products of the reaction pathway. The circular forms occur at very low levels in conjugating cells, but are stable. Sequencing analysis showed that many of the circles (as many as 50% of those examined) reflected a precise deletion in the M and R regions. The remaining circles were either smaller or larger and contained varying lengths of sequences derived from the chromosomal DNA surrounding the eliminated region. The chromosomal junctions left behind after deletion were more precise, although deletions in either the M or R regions can generate any of several alternative junctions (1). Some new chromosomal junctions were detected in the present study. The results suggest that the deleted segment is released as a linear DNA species that is degraded rapidly. The species is only rarely converted to the stable circles we detect. The deletion mechanism is different from those proposed for deletion events in hypotrichous ciliates (2-4), and does not reflect a conservative site-specific recombination process such as that promoted by the bacteriophage lambda integrase (5).     

Formation and Fate of Extranuclear Chromatin Bodies in Tetrahymena

The fate of extranuclear chromatin bodies (ECBs) formed by exclusion of macronuclear material at the time of karyokinesis was followed quantitatively in Tetrahymena pyriformis strain GL-I. In a logarithmic growth phase culture, 51% of the dividing cells produced one (43%) or more (8%) ECBs. Most of these gradually disappear before the next cell division, but ? 13% are retained and carried into subsequent cell cycles. The random distribution of ECBs into anterior or posterior daughter cells, their staining and morphological characteristics, and their rapid loss in cells in starvation medium, all indicate that ECBs play no more of a role in cellular activity than that of an internally produced food vacuole.     

Preparation of chromatin containing ribosomal deoxyribonucleic acid from the macronucleus of Tetrahymena pyriformis.

A method is described that enables a chromatin fraction containing ribosomal DNA (DNA containing sequences coding for rRNA) to be prepared from the macronuclei of growing or stationary cultures of Tetrahymena pyriformis. This material is obtained in yields of between 25 and 75% of the theoretical maximum. The DNA in this fraction was identified as ribosomal DNA on the basis of its density and molecular weight, and it appears not to be appreciably contaminated by other DNA. The method relies on the approximate assumption that ribosomal DNA is the smallest species of DNA in chromatin in the nucleus, and avoids the use of mechanical force, or enzyme action, to fractionate chromatin.     

Visualization of chromatin substructure: upsilon bodies

Spread chromatin fibers, from isolated eucaryotic nuclei, reveal linear arrays of spherical particles (upsilon bodies), about 70 A in diameter, connected by thin filaments about 15 A wide. These particles have been observed in freshly isolated nuclei from rat thymus, rat liver, and chicken erythrocytes. In addition, upsilon bodies can be visualized in preparations of isolated sheared chromatin, and in chromatin reconstructed from dissociating solvent conditions (i.e., high urea-NaCl concentration). As a criterion for perturbation of native chromatin structure low-angle X-ray diffraction patterns were obtained from nuclear pellets at different stages in the preparation of nuclei fro electron microscopy. These results suggest that the particulate (upsilon body) structures observed by electron microscopy may be closely related to the native configuration of chromatin.     

Analysis of chromatin repeat units in logarithmically and stationary growing cells of Paramecium aurelia and Tetrahymena pyriformis.

We have used micrococcal nuclease as a probe of the repeating structure of chromatin isolated from the macronuclei of logarithmically and stationary grown $\text{Paramecium aurelia}$ and $\text{Tetrahymena pyriformis}$. For both these lower eukaryotes, the monomer size is shown to vary depending on the stage in the growth cycle. $\text{P. aurelia}$ exhibits a monomer size of 153±7 bp and 178±6 bp and $\text{T. pyriformis}$ 207±10 bp and 230±10 bp in logarithmic and stationary cells, respectively. Both exhibit a nucleosome size of 140 bp. We discuss the possible association of these changes with histone content and nuclear activity changes, and also a possible reason for the divergence from the size pattern of monomer repeats seen in lower eukaryotes by $\text{T. pyriformis}$.     

The Formation and Fate of Tetrahymena patula Cryptostomes

A reliable method for producing reproductive cysts in Tetrahymena patula is described. The procedure involves the isolation of macrostomes without cytopharyngeal pouches in microdrops of distilled water under oil. The study of silver-impregnated specimens has shown that a complex pattern of oral resorption and reformation occurs within the cyst that leads to the formation of a group of small cells with recessed oral apparatuses. These cells, called “cryptostomes,” swim very rapidly on excystment and are incapable of either feeding or reproducing. They are presumably dispersal forms. Oral morphogenesis during the transformation of excysted cryptostomes into microstomes and macrostomes is also described.     

Changes in Oral Apparatus Structure Accompanying Vacuolar Formation in the Macrostomal Form of Tetrahymena vorax. A Model for the Formation of Food Vacuoles in Tetrahymena1

The large cytopharyngeal pouch of the macrostomal form of Tetrahymena vorax, following the addition of calcium, can form a sealed, empty vacuole. The open cytostomal region of this cell, which averages about 16 μ in diameter, is closed by an upward (ventral) movement of the right and posterior ribbed walls, both of which project into the cytostomal cavity. At the same time, the anterior and left walls of the cytostome-cytopharyngeal complex move to the right, forming a diagonally (right to left) placed furrow in the floor of the buccal cavity as these walls meet. As a result of the movement, the edges of the single membrane-bounded cytopharyngeal pouch are brought together and fuse, producing the closed vacuole. Elements of the cytoskeleton appear to participate in the closure process. Three major groups of ribbed wall microtubules support the open cytostome. The anterior ribbed wall microtubules pass laterally along the anterior (dorsal) portion of the cytopharyngeal pouch to the left where they end in the specialized cytoplasm. Middle oral rib microtubules terminate at the right and posterior margin of the cytopharynx while microtubules from the most posterior region of the ribbed wall pass to the left terminating in the specialized cytoplasm. The fine filamentous reticulum, a striated reticulum that borders the right, posterior, and anterior margins of the cytostome-cytopharyngeal complex, is in an ideal position to participate in these movements. It is anchored anteriorly high up in the buccal cavity to the cross-connective between the third membranelle and the undulating membrane complex. It courses beneath the right and posterior ribbed walls and runs laterally along the anterior margin of the cytopharynx to the left side. Contraction or pulling of this reticulum would act to bring the microtubule-reinforced walls of the cytopharynx together permitting fusion of the cytopharyngeal pouch membranes to form a sealed vacuole.