The nucleolar organizer of Plethodon cinereus cinereus (Green)

The lampbrush nucleolar organizers in P. c. cinereus are on the shorter arms of the 7th longest bivalent, near to the centromeres. The organizer consists of a non-chromomeric, Feulgen-negative section of the half bivalent axis inserted between typically chromomeric regions with normal lampbrush loops. The main axis of the organizer may appear double or single. The distributions and lengths of the double regions are variable. The axial strand of the organizer can be broken with DNase. Nucleolus-like objects are attached at irregular intervals along the organizer axis. The attached nucleoli closely resemble the free nucleoli in the same nucleus. Where free and attached nucleoli appear as beaded rings, the general characteristics and range of lengths of the attached rings are similar to those of the free rings. The attached rings occur singly, in pairs, or in clusters. The point of attachment of a ring is sometimes marked by a granule on the organizer axis. Pairs of attached nucleolar rings sometimes form double bridges in which the nucleoli extend linearly across a gap in the organizer axis. The length of the organizer varies from 20 μm to 300 μm, depending, at least in part, on the stage of oocyte development. The difference is a function of the length of the axial strand. Nucleolar organizers similar to that described for P. c. cinereus have been seen on the lampbrush bivalents of 7 other species of Plethodon and 2 species of Eurycea. In all of these, the 2 organizers of the nucleolus bivalents differ in length.     

The nucleolar organizer of Plethodon cinereus cinereus (Green)

The 7th longest lampbrush bivalent in oocytes of Plethodon cinereus cinereus has a region of attached oocyte nucleoli near to the centromere on the shorter arm of each half bivalent. When squash preparations of P. c. cinereus spermatocytes are treated with NaOH to denature chromosomal DNA, and subsequently incubated in a solution of (³H) ribosomal RNA from Xenopus cell cultures, the ribosomal RNA binds specifically to a region near the centromeres on the shorter arms of the 7th longest bivalent, and to a region near the end of the shorter arm of the 14th bivalent. The amount of ribosomal RNA bound to the 7th bivalent at diplotene and 1st meiotic metaphase is regularly different on the 2 halves of the bivalent. Each half of the 14th bivalent is usually labelled more heavily than the less heavily labelled half of the 7th bivalent. These observations are discussed in relation to the involvement of nucleolar organizers in gene amplification and rectification.     

Development of the large nucleolus in the oocytes of the copepod Acanthocyclops vernalis: an electron microscope study

A central feature of oogenesis in the copepod crustacean, Acanthocyclops vernalis, is the development of a very large nucleolus in the oocytes. This nucleolus appears to be the only source of rRNA for the oocyte, as no helper cells are present. Previous work has suggested that ribosomal DNA sequences other than those found at the morphological nucleolar organizers are participating in the elaboration of this nucleolus. It has been hypothesized that chromatin diminution, which occurs during early embryonic development, may involve the loss of these rDNA sequences, which are needed only for the production of ribosomes during oogenesis. The present study examines the development of the large oocyte nucleolus at the electron microscopic level. Nucleologenesis in A. vernalis was found to proceed through 5 stages. During the first 3 stages nucleolar morphology resembled that described in other organisms. In the last 2, however, nucleolar morphology changed radically and the nucleolus was seen to increase greatly in size while breaking up into multiple subunits. The subunits initially resemble active nucleoli, although in the last stage, synthesis appears to stop, as the nucleolus was found to consist only of dense areas containing ribosome-like particles. These observations are consistent with the hypothesis that diminuted DNA contains ribosomal RNA genes.     

Variation in the activity of nucleolar organizers and their ribosomal gene content

In metaphase preparations from leucocytes of the toad, Bufo marinus, conspicuous secondary constrictions are present in the number 7 pair of chromosomes. These constrictions were considered to be the nucleolar organizers since they were associated with nucleoli during prophase. In 35 out of 60 individuals taken from natural populations, the homologous nucleolar organizers produced two equal-sized nucleoli and secondary constrictions (Group I animals). Pour animals (Group II) had only one very large secondary constriction in the majority of their metaphase preparations and an abnormally high frequency of cells containing one nucleolus. The remaining 21 animals (Group III) had unequal-sized constrictions in most of their metaphases but were more variable than the individuals of Groups I or II since they also had metaphases with two equal constrictions or only one constriction.The DNA from individuals of each group was hybridized with radioactive ribosomal RNA in order to correlate the size of nucleoli and constrictions with the amount of DNA (rDNA) homologous to ribosomal RNA. The two animals of Group II which were studied contained 0.056% of their genome homologous to ribosomal RNA a value considerably higher than those found for any of the animals of Groups I or III. These high values for rDNA coupled with the morphological appearance of the nucleolar homologues suggested a duplication of the nucleolar organizer in the homologue with the long constriction. The amount of rDNA in animals of Group I and III varied between 0.025 and 0.048% of the genome. Although the animals with unequal-sized constrictions (Group III) had generally lower contents of rDNA than those with equal-sized constrictions (Group I), the values overlapped between the two groups. Further evidence which correlates the size of nucleoli with the number of ribosomal RNA genes comes from studies with a small nucleolar mutant of the Mexican axolotl (Ambystoma mexicanum). Animals homozygous for this deletion were found to contain only 55% of the complement of rDNA present in the wild type. It is concluded that partial deletions and duplications of the nucleolar organizer as well as highly variable contents of rDNA are common in the genome of these amphibians.Presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Biology Department, University of Rochester.     

Patterns of activity of nucleolar organizer regions during spermatogenesis and oogenesis in Kalotermes flavicollis Fabr. (Insecta: Isoptera) analyzed by silver staining

The distribution and the behaviour of the nucleolus organizer regions (NORs) were analysed during the spermatogenesis and oogenesis of K. flavicollis with the silver staining method. The Ag-stainability of the NORs increases in growing spermatocytes up to pachytene and is absent during the remainder of the meiotic prophase. During female meiosis the nucleolar material undergoes a more complex transformation. It is active until pachytene; in early diplotene the mass of silver stainable material progressively increases as an effect of rDNA amplification. By the end of meiotic prophase the nucleolar strands disappear and a large nucleolus is rebuilt in the mature oocyte.     

Ribosomal gene amplification does not occur in the oocytes of Locusta migratoria

It has been suggested that Locusta migratoria amplifies its ribosomal RNA genes in the growing oocytes (Kunz (1967) Chromosoma20, 332–370). Cloned ribosomal DNA of L. migratoria was used to analyze rDNA structure and number. The rDNA is localized on three chromosome pairs in six nucleolus organizers. It was found that all structural variants of the rRNA genes which have been described previously are represented in the same relative amounts in DNA from isolated oocytes as in somatic cells. Furthermore, the rRNA gene number is not increased in oocyte DNA, i.e., amplification does not occur. Therefore, the large number of multiple nucleoli seen in the growing oocytes has to be interpreted as the fully extended and fully active set of chromosomal rRNA genes. The total rRNA gene number was determined by dot blot hybridization to be about 3300 genes/haploid genome.     

Multiplication of nucleolar fibrillar centres and absence of rDNA amplification in mouse ooctye during meiotic prophase I

During meiotic prophase I the nucleolus of the mouse oocyte assumes a reticulate structure of ‘nucleolonema’ type. This change coincides with the appearance of several secondary fibrillar centres. The number of these centres at diplotene (97–113), largely exceeds that of nucleolar organizers (4c DNA = 20 NORs). The quantitatative analysis of autoradiographs after hybridization in situ with -³H-uridine labelled rRNA, enabled us to demonstrate that the multiplication of the fibrillar centres in mouse oocyte nucleolus during meiotic prophase I is not the result of an amplification of the rDNA. The number of silver grains in pachytene and diplotene nuclei was twice that counted for somatic cell and oogonium nuclei (2c DNA).     

Isolation and characterization of different-sized nucleoli from Ehrlich ascites tumor cells : Evidence of different nucleolar ribosomal cistrons

A procedure was developed for isolation of variously sized nucleoli in order to study the mechanism of nucleolar formation from multiple nucleolar organizers and to compare the compositions of different-sized nucleoli from Ehrlich ascites tumor cells. Relatively small nucleoli and large nucleoli from Ehrlich ascites tumor cells were separated by centrifugation at 400 g for 5 min in a layer of 0.34 M sucrose over 0.88 M sucrose. Small nucleoli remained in the 0.34 M sucrose layer while the large nucleoli accumulated in the 0.88 M sucrose.Three fractions, provisionally named small, intermediate and large nucleoli, containing 0.33, 0.41 and 0.84 pg DNA/nucleolus, respectively, were separated. Unfractionated nucleoli contained 0.59 pg DNA/nucleolus. The RNA content also increased with the size of the nucleolus and no significant difference was observed in the RNA/DNA ratios in the three fractions. Large nucleoli incorporated more [³H]uridine and [³²P]orthophosphate into RNA than did small nucleoli, but the base compositions of the RNAs extracted from the different-sized nucleoli were similar. No significant fragmentation occurred on sonication of large nucleoli for 3 min, so the observed difference in the DNA contents was not due to mechanical damage of the nucleoli.The DNAs of these different-sized nucleoli were analysed on CsCl gradients. The nucleoli contained similar percentages of satellite DNA (20–22%) which were also similar to those of total, unfractionated nucleoli. Approx. 10% of the extranucleolar DNA is satellite DNA—thus the nucleolar fractions were probably not appreciably contaminated with extranucleolar DNA. The DNA of small nucleoli contained a slightly lower percentage (0.058%) of ribosomal cistrons than large nucleoli (0.081%). This means that the higher content of DNA in the large nucleoli is not merely due to longer sized chromatin with extra regions of the vicinity of nucleolar organizers. Thus these results suggest that the total content of ribosomal cistrons/nucleolus is roughly proportional to the DNA content of the nucleoli, at least in Ehrlich ascites tumor cells. Namely, the number of ribosomal cistrons per nucleolus for small, intermediate and large nucleoli is 40, 60 and 130, respectively.     

An investigation of some problems concerning nucleolus organizers in salamanders

Observed differences in the sizes of lampbrush nucleolus organizers in Plethodon cinereus have been shown by in situ hybridization to reflect true molecular differences in the numbers of ribosomal cistrons located at these organizers. Likewise, from in situ hybridization experiments on lampbrush and spermatocyte chromosomes it has been shown that animals may be, and indeed usually are, heterozygous with respect to the numbers of ribosomal cistrons on each half of the nucleolus bivalent. Filter hybridizations carried out on 33 males from a New Jersey population and 20 males from a Connecticut population have shown a 7.5-fold range in the numbers of ribosomal cistrons per diploid cell in the New Jersey population, and a 2.5-fold range in the Connecticut population. In view of the general heterozygosity of nucleolus organizers in these animals, the actual range in nucleolus organizer sizes in the New Jersey population is estimated to be at least 15-fold.     

Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.     

Absence of rDNA amplification in the uninucleolate oocyte of the cockroach Blattella germanica (Oorthoptera: Blattidae)

Amplification of the genes coding for ribosomal RNA oocurs in the oocytes of a wide variety of organisms. In oocytes of various species of crickets (Orthoptera: Gryllidae) the amplified DNA is contained in a large extrachromosomal DNA body. Multiple nucleoli form about the periphery of the DNA body during the diplotene stage of meiosis I. In contrast to the general pattern of orthopteran oocytes, oocytes of the cockroach Blattella germanica demonstrate a single large nucleolus instead of many nucleoli. In order to determine whether the genes coding for rRNA are amplified in the oocytes of B. germanica, the relative amount of rDNA in oocytes was compared with the rDNA content of spermatocytes and somatic cells. An extrachromosomal DNA body similar to that present in crickets is not present in B. germanica. A satellite DNA band which contains nucleotide sequences complementary to rRNA accounts for approximately 3-5% of the total DNA in somatic and in male and female gametogenic tissues. Female cells contain approximately twice as much rDNA as do male cells. An XX-XO sex-determining mechanism is operative in B. germanica. In situ hybridization with rRNA indicates that the nucleolar organizer is located on one end of the X chromosome and that oocytes do not contain more than twice the amount of rDNA found in spermato cytes. The data indicate that rDNA is not amplified in the uninucleolate oocyte of B germanica.     

Nucleolus-like bodies in micronuclei of cultured Xenopus cells

Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeply-stained nucleoli incorporated [³H]uridine and appeared by electron microscopy to have typical nucleolar morphology with fibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.     

The development of a large nucleolus during oogenesis in Acanthocyclops vernalis (Crustacea, Copepoda) and its possible relationship to chromatin diminution

The development of a single, very large (25-35 microns diameter) nucleolus during oogenesis in the crustacean Acanthocyclops vernalis is described. The nucleolus is the site of ribosomal RNA production in the egg, as shown by in situ hybridization, and apparently the only source, as accessory cells are not observed. Ribosomal DNA amplification, as manifested by the presence of multiple nucleoli, is also not observed. Silver staining and C-banding suggest that chromosomal regions other than the nucleolar organizer are involved with the elaboration of the nucleolus. These observations, along with what is known about the nature of the DNA lost during the developmental process of chromatin diminution in this organism, suggest a relationship between the large oocyte nucleolus and the DNA lost during diminution.     

Organization of argyrophilic nucleolar material throughout the division cycle of meristematic cells

Summary The silver impregnation of nucleolar material facilitated the study of the morphological changes which take place in the nucleolus throughout the division cycle in root tip cells ofAllium cepa. The nucleolus appears to undergo no morphological changes throughout the interphase. It undergoes disorganization during the prophase, while in the telophase it appears uniformly on the chromatin as condensing into prenucleolar bodies.The appearance of the prenucleolar bodies is unaffected by puromycin, cordycepin, or ethidium bromide. This suggests that the argyrophilic material does not undergo synthesis during the telophase, nor require RNA or protein synthesis to effect the aggregation into prenucleolar bodies. However, the organization of nucleoli from prenucleolar bodies is inhibited by both cordycepin and ethidium bromide, suggesting that RNA synthesis is involved in this proccess.In aneuploid nuclei induced by treatment with colchicine we observed the appearance of prenucleolar bodies during the telophase even in the absence of the nucleolar organizer, but in this case the formation of nucleoli fails to take place. The nucleolar organizers proved to be capable of acting only in the nucleus to which they belong, but not on other nuclei within the same cytoplasm belonging to multinucleate cells.It seems logical to assume that one of the roles of the nucleolar organizer is related with the above-mentioned RNA synthesis, which is required to the aggregation of prenucleolar bodies into nucleoli.The work reported in the paper was undertaken during the tenure of a Research Training Fellowship awarded by the International Agency for Research on Cancer.