Thecamoeba onigiri n. sp. (Amoebozoa, Discosea, Thecamoebida) – one more species of the genus Thecamoeba with polymorphic nuclear structure

The structure of a "noncanonical" nucleolus of vitellogenic oocytes in the sea urchin Paracentrotus lividus was studied using the inhibitor of transcription actinomycin D. In the control cells, the nucleolus consists of two separated structural subdomains: the dense fibrillar-granular peripheral area and the fibrillar central area. The nucleolus did not contain subdomains corresponding to the fibrillar center and dense fibrillar component of "typical" nucleoli. After treatment with actinomycin D, numerous argyrophilic granules appeared in the karyoplasm, the intranucleolar DNA became compact, and the nucleolar material was segregated into two or three separated zones, the residual peripheral area being the densest and largest. Lesser zones had a decreased electron density and contained argyrophilic proteins and, apparently, the nucleolar organizer material. These results suggest that, for normal rRNA expression and processing, the presence of structural subdomains in the nucleolus, such as fibrillar complexes and a dense fibrillar component, is not essential.

Dominance of a 5-azacytidine-substituted nucleolar organizer region (AZA NOR) over another with normal DNA when confined to a common nucleus or to different nuclei in a common cell was studied in Allium cepa L. root meristems. AZA administration took place for one S period. The assessment of NOR dominance was done either in early Gi (nucleologen-esis) or later in interphase (mature nucleoli). At the mitosis immediately preceding the second interphase after AZA, the segregated AZA and normal chromatids of each chromosome of the pair of homologues were confined to a single binucleate cell by inhibiting the formation of the cell plate. Anaphase segregation of the homologous AZA NORs was seen to occur at random. The AZA NORs completed nucleologenesis after a shorter time than normal NORs, independently of the intranuclear or intracellular presence of other NORs. After nucleologenesis was over in the AZA NORs, a few of the normal NORs, when in a common nucleus, remained permanently inefficient in developing a full-size mature nucleolus or any nucleolus at all (partial or total dominance, respectively). Dominance of an AZA NOR over its sister or allelic NOR with normal DNA led to the new nucleolar patterns of asymmetry and inverse symmetry, respectively, making their appearance in these binucleate cells.

The nucleolar-like bodies or micronucleoli of Sciara coprophila salivary gland nuclei have been studied with phase contrast, the Nomarski optics, Azure B staining, and electron microscopy. In the late fourth instar the main nucleolus formed at the X-chromosome may become extensively fragmented and may appear as a large aggregate of micronucleoli. At about the same time large numbers of micronucleoli in a more peripheral location are also found. Studies in partially squashed and stained nuclei, as well as in unfixed glands have shown that, at a time when the nucleolar material is abundant, the X-NOR is highly ramified with its branches permeating much of the nuclear space. These observations make it appear probable that most or all of the nucleolar material, even the more peripherally located, is actually in contact with the main nucleolar organizer or its branches. On the other hand, many chromosomal bands are also in close association with micronucleoli. At the level of electron microscopy some of the associations between chromo somal bands and micronucleoli are very intimate with the nucleolar material often found deep within the band. In other instances there seems to be physical continuity between extensions of band chromatin and certain areas of the fibrillar component. The bands in question could be the sites of secondary nucleolar organizers. In the electron microscope a large aggregate of micronucleoli, interspersed with portions of chromatin can often be seen in an approximately central location. This is interpreted as the main nucleolus with portions of its NOR. Both the main nucleolus and the more peripheral micronucleoli are indistinguishable in their fine structure and show the components typically found in nucleoli, i.e., fibrils and granules. On the other hand the fine structure of both RNA and DNA puffs is strikingly different.

Individual organisms of Amoeba proteus have been fixed in buffered osmium tetroxide in either 0.9 per cent NaCl or 0.01 per cent CaCl2, sectioned, and studied in the electron microscope in interphase and in several stages of mitosis. The helices typical of interphase nuclei do not coexist with condensed chromatin and thus either represent a DNA configuration unique to interphase or are not DNA at all. The membranes of the complex nuclear envelope are present in all stages observed but are discontinuous in metaphase. The inner, thick, honeycomb layer of the nuclear envelope disappears during prophase, reappearing after telophase when nuclear reconstruction is in progress. Nucleoli decrease in size and number during prophase and re-form during telophase in association with the chromatin network. In the early reconstruction nucleus, the nucleolar material forms into thin, sheet-like configurations which are closely associated with small amounts of chromatin and are closely applied to the inner, partially formed layer of the nuclear envelope. It is proposed that nucleolar material is implicated in the formation of the inner layer of the envelope and that there is a configuration of nucleolar material peculiar to this time. The plasmalemma is partially denuded of its fringe-like material during division.

Influence of the nucleolus on DNA synthesis and mitosis in Vicia faba

Frequencies of Circular Units of Nucleolar DNA in Oocytes of Two Insects, Acheta domesticus and Dytiscus marginalis, and Changes of Nucleolar Morphology during Oogenesis

Marine and freshwater populations of the red alga Bangia were sampled throughout most of the known geographic range in North America. Marine samples were obtained from the Pacific and Atlantic coasts, central Arctic, Gulf of Mexico, and the Virgin Islands. Freshwater samples came from populations in the Laurentian Great Lakes, St. Lawrence River, and Lake Simcoe. In addition, specimens from freshwater populations from Italy, England, and Ireland were included for comparison. DNA sequence analyses of the Rubisco spacer, rbcL gene, and 18S rRNA gene, plus morphometric analysis, were used to discern possible biogeographic and taxonomic trends. No correlation was detected between the molecular and morphological analyses; however, some trends were consistent among the three molecular analyses. For example, all the freshwater collections were nearly identical, indicating that a single lineage may exist in freshwater systems. In addition, the Virgin Islands sample was distinct from all others in both the rbcL and 18S rRNA gene sequences, but quite similar in morphology to those collections with small filament diameter. The Alaska sample clustered consistently with a primarily Atlantic clade with few sequence differences. This finding may indicate a transarctic invasion. Sequence analysis of the 18S rRNA gene yielded a previously unreported intron (in Bangia) located between helices 21 and 20′. All freshwater collections contained this intron, but it was present only in some marine collections. Considerable sequence divergence occurred among groups of collections in both the rbcL (0–16%) and 18S rRNA (0–10.6%) genes with little corresponding diversity in morphology. These values are higher than those reported previously for intergeneric, intrafamilial, or interfamilial comparisons in higher red algal taxa, but are consistent with those reported for the members of the Bangiales. Sequence analysis of the 18S rRNA gene of Bangia collections with six Porphyra species yielded a tree in which Porphyra is paraphyletic within Bangia. In addition, the molecular divergence appears to be quite low between the two genera.

The evolution of the chromosomal location of ribosomal RNA gene clusters and the organization of heterochromatin in the Drosophila melanogaster group were investigated using fluorescence in situ hybridization and DAPI staining to mitotic chromosomes. The investigation of 18 species (11 of which were being examined for the first time) belonging to the melanogaster and ananassae subgroups suggests that the ancestral configuration consists of one nucleolus organizer (NOR) on each sex chromosome. This pattern, which is conserved throughout the melanogaster subgroup, except in D. simulans and D. sechellia, was observed only in the ercepeae complex within the ananassae subgroup. Both sex-linked NORs must have been lost in the lineage leading to D. varians and in the ananassae and bipectinata complexes, whereas new sites, characterized by intra-species variation in hybridization signal size, appeared on the fourth chromosome related to heterochromatic rearrangements. Nucleolar material is thought to be required for sex chromosome pairing and disjunction in a variety of organisms including Drosophila. Thus, either remnant sequences, possibly intergenic spacer repeats, are still present in the sex chromosomes which have lost their NORs (as observed in D. simulans and D. sechellia), or an alternative mechanism has evolved.

The above account would clearly indicate that in connection with the meiotic cytology ofDaemia extensa, there are a number of points of special interest such as the intranuclear origin of the achromatic figure, a well-defined period of rest during interkinesis, deposition of the granules at the equatorial region leading to cytokinesis and a very significant role of the nucleolus. The structure of the nucleolus in the resting condition of the microspore mother-cell and its subsequent behaviour during the two meiotic divisions establish beyond doubt that it is a storehouse for chromatin and that there exists a specific relationship between the nucleolar material and the cyclic alterations of chromosomes. It is further inferred that there is a definite correspondence between the number of chromatin pieces of which the nucleolus is composed and the haploid number of chromosomesi. e. 12. It is not possible to give in this note, the details of evidence in this direction nor the discussion that the subject really deserves. In a recent review on nucleoli and the related nuclear structures, Gates4 has given an exhaustive account of the subject in all its aspects and has quoted an extensive literature at the end. Some of the most notable workers whose cytological investigations have thrown a great deal of light on the origin and history of the nucleolus are Bhatia,1 van Camp,2 Gates,3 Heitz,5 Latter,8 Ludford,7 McClintock,8 Nandi,9 Nawashin10 and Zirkle.11 Of particular interest in connection with the present investigation are the findings of van Camp2 and Nandi.9 The former was of opinion that the nucleoli originated from the chromosomes at telophase in the form of small globules which later by fusion formed one large nucleolus. According to the latter the nucleolar material got liberated during the heterotypic telophase as small globules which, under the influence of the nucleolar body present in one chromosome of the haploid complement, were organised into a definite nucleolus that remained associated with one particular chromosome. In the case ofDaemia extensa of which the account has been given above, each haploid chromosome gave rise to a deeply-stained chromatin piece during the heterotypic telophase so that ultimately there originated 12 such pieces and these coalesced together to form one nucleolus during interphase. A detailed account of all the facts would be published later.

Hela cells were impregnated with silver according to Paweletz et al. (1967). In cells in mitosis not only the nucleolar organizer regions (NORs) are strongly impregnated but also part of the nucleolar material, which accumulates in and around the chromosomes. The treatment with adenosine, which in interphase cells spreads the nucleolar material within the nucleus, also distributes the argentophilic material in and around the chromosomes. During the reconstruction phase this material reassembles around the NORs to form parts of the new nucleolus. The silver impregnation technique clearly demonstrates that two main components are responsible for the argentophily of the nucleolus. This is in agreement with the results obtained by Lischwe et al. (1979).

Fine structure of nucleoli in micronucleated cells

The process of nucleolus formation has been studied by electron microscopy in spermatogonia of new-born, 15-day-old mice. One of two heteropycnotic sex chromosomes is concerned with nucleolus formation in the type A spermatogonia. The evidence for such formation has been presented with regard to behaviour and fine structure of both sex chromosome and nucleolus, Nucleolar material appears at one of two heteropycnotic sex chromosomes which are closely attached to the nuclear envelope. The two sex chromosomes approach each other, and subsequently one of them migrates into the central part of the nucleoplasm, being related to the nucleolar material which develops to show a nucleolar configuration. The sex chromosomes are homogeneously electron dense during the nucleolus formation, but assume a vesicular form at the middle stage of its development. The nucleolus is mostly of fibrillar and amorphous components at early stages of its development, but the granular components increases in amount as development proceeds. The final, mature nucleolus is composed of irregularly twisted nucleolonemata consisting of granular components, separated from fibrillar and amorphous areas. The compactly dense sex chromosome remains closely connected with the mature nucleolus.

The process of nucleolus formation has been studied by electron microscopy in spermatogonia of new-born, 15-day-old mice. One of two heteropycnotic sex chromosomes is concerned with nucleolus formation in the type A spermatogonia. The evidence for such formation has been presented with regard to behaviour and fine structure of both sex chromosome and nucleolus, Nucleolar material appears at one of two heteropycnotic sex chromosomes which are closely attached to the nuclear envelope. The two sex chromosomes approach each other, and subsequently one of them migrates into the central part of the nucleoplasm, being related to the nucleolar material which develops to show a nucleolar configuration. The sex chromosomes are homogeneously electron dense during the nucleolus formation, but assume a vesicular form at the middle stage of its development. The nucleolus is mostly of fibrillar and amorphous components at early stages of its development, but the granular components increases in amount as development proceeds. The final, mature nucleolus is composed of irregularly twisted nucleolonemata consisting of granular components, separated from fibrillar and amorphous areas. The compactly dense sex chromosome remains closely connected with the mature nucleolus.

The locations of the ribosomal DNA (rDNA) insertion elements type I and type II along the polytene chromosomes of three Drosophila species of the melanogaster subgroup--D. simulans, D. mauritiana and D. melanogaster--have been compared. In situ hybridization has shown that the intragenomic distribution of type I as well as of type II insertions is different for these related species. In particular, we have revealed rDNA-free autosomal sites, containing type II element sequences within the D. simulans and D. mauritiana chromosomes. This finding confirms the ability of this type of insertion to transpose, as was demonstrated earlier for Bombyx mori. The appearance of the rDNA not associated with the nucleolar organizers, evident by additional nucleoli, occurred with species-specific frequency. At the same time, for all three species the pattern of such changes (an attachment of the nucleoli to varying sites of the chromosomes and the presence of ectopic contacts between them, a composition of the rDNA repeats in the nucleolar material not integrated at the nucleolar organizer) was similar. The number of additional nucleoli in the hybrid polytene nuclei corresponded to the value of the parental species exhibiting nucleolar replicative dominance.

PHASE-CONTRAST observation and electron microscopy indicate that the nucleolus is structurally heterogeneous consisting of a ground material in which is embedded a more compactly aggregated material generally described as filamentous (Estable and Sotelo1 proposed for them the names of ‘pars amorpha’ and ‘nucleolonema’). Nucleoli of plant and animal cells are seen at mitosis and meiosis associated with a particular chromosome or chromosomes—the so-called ‘nucleolar organizer’ chromosomes—at the site of a heterochromatic region. If a chromosomal deletion includes such a region or if a mutation occurs2, nucleolar material is then produced at other ‘loci’ (the latent organizers).