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.     

Cytogenetics of Crotalaria V. Supernumerary nucleoli in C. agatiflora (Leguminosae)

Of 35 species of Crotalaria (Leguminosae) studied, all but one had usually one nucleolus in the premetaphase I cells. In C. agatiflora 300 PMCs from four out of five plants were investigated and the percentage cells with more than one nucleolus was determined as well as the nature of nucleolar attachment to bivalents and the range and the sizes of various nucleoli present in the cell. The aberrant cells ranged from 42 to 50%. The nucleoli (1–6) were usually attached to different bivalents. Where one nucleolus was present in the cell, it was always attached to an X-shaped bivalent, formed of a pair of nucleolar chromosomes. In the aberrant cells one nucleolus usually was attached to such a bivalent. The presence of accessory nucleoli has been attributed to hybridity as a result of large-scale intercrossing among five subspecies and consequent dispersal of intermediates in the adjoining areas where C. agatiflora grows wild (East Africa and Ethiopia). The change in the regulatory system of the cell caused by hybridity results in activation of latent nucleolar organizers, although their overall presence in the genome is not due to hybridity.     

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.     

Number and Nuclear Localisation of Nucleoli in Mammalian Spermatocytes

In seven mammalian species, including man, the position and number of nucleoli in pachytene spermatocyte nuclei were studied from electron microscope (EM) nuclear sections or bivalent microspreads. The number and position of the nucleolar organiser regions (NORs) in mitotic and meiotic chromosomes were also analysed, using silver staining techniques and in situ hybridisation protocols. The general organisation of pachytene spermatocyte nucleoli was almost the same, with only minor morphological differences between species. The terminal NORs of Thylamys elegans (Didelphoidea, Marsupialia), Dromiciops gliroides (Microbiotheridae, Marsupialia), Phyllotys osgoodi (Rodentia, Muridae) and man, always gave rise to peripheral nucleoli in the spermatocyte nucleus. In turn, the intercalated NORs from Octodon degus, Ctenomys opimus (Rodentia, Octodontidae) and Chinchilla lanigera (Rodentia, Cavidae), gave rise to central nucleoli. In species with a single nucleolar bivalent, just one nucleolus is formed, while in those with multiple nucleolar bivalents a variable number of nucleoli are formed by association of different nucleolar bivalents or NORs that occupy the same nuclear peripheral space (Phyllotis and man). It can be concluded that the position of each nucleolus within the spermatocyte nucleus is mainly dependent upon: (1) the position of the NOR in the nucleolar bivalent synaptonemal complex (SC), (2) the nuclear pathway of the nucleolar bivalent SC, being both telomeric ends attached to the nuclear envelope, and (3) the association between nucleolar bivalents by means of their NOR-nucleolar domains that occupy the same nuclear space. Thus, the distribution of nucleoli within the nuclear space of spermatocytes is non-random and it is consistent with the existence of a species-specific meiotic nuclear architecture.     

The chromosomal localization of a heavy satellite DNA in the testis of Plethodon c. cinereus

When DNA from blood or liver of Plethodon c. cinereus is centrifuged to equilibrium in cesium chloride it separates out into 2 components. The smaller or satellite component is relatively rich in G + C and is therefore heavy, and it amounts to about 2% of the total DNA. The heavy satellite does not include the ribosomal cistrons, and it is unrelated to the nucleolar organizer. When squash preparations of cells from the testis of P. c. cinereus are incubated in synthetic E³RNA complementary to the satellite DNA, the RNA anneals specifically to the centromeric heterochromatin of spermatogonia, spermatocytes, and spermatids, and to the centromeric regions of all discernible chromosomes. RNA/DNA hybrids were located by autoradiography. H³RNA complementary to the major component of the DNA anneals to all nuclei and to all parts of the chromosomes. H³RNA complementary to nucleolar DNA from Xenopus laevis anneals specifically to the chromatin associated with nucleoli in nuclei at various stages of the meiotic divisions. The nature of the centromeric heterochromatin and its role in the meiotic divisions are discussed.     

A fresh look at meiosis and centromeric heterochromatin in the red-backed salamander,Plethodon cinereus cinereus (Green)

Male meiosis, with special regard to the centromeric heterochromatin and to centromeric structure, has been studied in the salamander, Plethodon cinereus cinereus. In this salamander, n = 14. Early meiotic prophase proceeds as described by other authors. Pachytene is followed by a diffuse stage in which much of the chromosomal DNA becomes reorganized into fine lateral loops which spring from the bivalent axes. These loops can be seen along the bivalent axes as early as zygotene. Loops are maximally extended in the diffuse stage. The formation of diplotene bivalents involves a return of this extended DNA into the axes of the bivalents. — At leptotone, centromeric heterochromatin is in one or a few large masses. These masses break up during zygotene. At pachytene there is one mass of heterochromatin at the centromeric region of each bivalent. The heterochromatin remains condensed in the diffuse stage. During diplotene, centromeric heterochromatin becomes less conspicuous, and it is possible to see 4 centromere granules in each diplotene bivalent. These observations support the view that centromeres replicate at pre-meiotic S-phase when the associated hetero-chromatin is replicated. In the interphase before the 2nd division, the hetero-chromatin often forms a broken ring corresponding to the positions of the centromeres at the end of anaphase 1. There are 14 masses of heterochromatin in nuclei at prophase of the 2nd division. In spermatids, the heterochromatin appears as a single solid mass or a broken ring.     

Lampenbürstenchromosomen und multiple Nukleolen bei Orthopteren

Isolated unfixed chromosomes from the oocytes of several grasshopper species, of Gryllus domesticus, and of two cockroaches have been investigated under phase contrast. As demonstrated previously in Locusta migratoria (Kunz, 1967), these chromosomes resemble the lampbrush chromosomes in amphibian oocytes. From these the lateral loops of the orthopteran chromosomes differ in that they are only one third as long (Fig. 3). The distinctness of chromomeres and chiasmata is considerably lower than that in amphibian oocytes (Fig. 4). — Besides the lampbrush chromosomes the oocyte nuclei of Orthoptera contain several hundred spheres or granules which are thought to be the multiple nucleoli (Fig. 6). In young oocytes, these nucleoli are vacuolated spheroids aggregated compactly in the center of the nucleus (Fig. 7). In the oocyte of the cricket, this center contains Feulgen-positive material which disappears in the early growth period when the nucleoli transform from solid structures to several hundred spheres. In oocytes of an intermediate size, both in the grasshoppers and Gryllus such spheroids are present. In the larger mature oocytes these spheres are localized peripherally around the nuclear envelope (Decticus; Fig. 10b), or the become extended into beaded ring forms (Gryllus; Fig. 12), or these rings are opened, stretched and connected in a row to form long “pearl-string” threads (Locusta, Acrida, Homorocoryphus). The spheroids around the nuclear envelope of Decticus look very similar to the solid and spheroidal nucleoli in young oocytes of the axolotl (Callan, 1966). The beaded rings of Gryllus resemble the ring-shaped nucleoli in the amphibian oocytes during their intermediate growing phase. — Following Keyl's (1966) hypothesis for the construction of replication units, these different appearances of multiple nucleoli are proposed to be results of a similar mode of extra-replication, but of different arrangement. In the case of Gryllus and Decticus the nucleolar DNA Anlagen are set free from the chromosomes, but they remain attached one behind the other in Locusta and some other grasshoppers.     

Nucleolar organizers and fibrillar centres in Triticum aestivum L., cv. Chinese spring

Serial sectioning of wheat roots prepared for electron microscopy was used to count the number of fibrillar centres per nucleus and nucleolus and to calculate their sizes. After growth at 35 ° C for two days the nucleoli became segregated and a one to one relationship was evident between chromosomal nucleolar organizers and fibrillar centres. This was confirmed using an aneuploid line carrying an additional pair of organizers. Quantitative studies showed that the fibrillar centres occupied a volume 0.24% of the total chromatin reticulum. From this it was calculated that only about 1/3 of the fibrillar centre material was likely to be nucleolar organizer chromatin. The other material was considered to be the protein revealed in silver staining studies. The importance of this was shown by its constant ratio to the size of all nucleoli in a given nucleus. — Evidence was found for the movement and fusion of organizer flanking regions during growth at 35 ° C. The number of junctions between chromatin and nucleolar organizers drops by about half giving one per organizer after segregation, and serial sectioning demonstrated such junctions in close proximity, an arrangement suggestive of incipient fusion.     

Non-relationship between nucleolus and sex chromosome system Xyp in Chelymorpha variabilis boheman (Coleoptera: Chrysomelidae)

Nucleolar-organizer region, nucleolus and mode of association of the sex bivalent were analyzed in spermatecytes of Chelymorpha variabilis Boheman. This species (2n=10II+Xy_p) shows the typical sex chromosome system of the group Polyphaga. The results of silver staining techniques showed the nucleolar organizer region localized in a subterminal position of an autosomal bivalent. During meiotic prophase the nucleolus was distinguished with the silver staining and acridine orange fluorescence technique up to diakinesis. The independence of nucleolus and sex bivalent Xy_p during meiosis is demonstrated. The positively silver staining but negatively orange-red material found within the parachute could be involved in the regular co-orientation of both sex chromosomes. After a longer hypotonic treatment, sex bivalents were observed elongated and paired only at one end during the pachytene stage. Along these sex chromosomes, C-bands showed positive blocks located in the pericentromeric and telomeric regions. Heterochromatic association of both sex chromosomes was suggested.     

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents

Summary Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.     

Funktionsstrukturen im Oocytenkern von Locusta migratoria

Examination of living oocyte nuclei of Locusta migratoria has revealed the presence of thread-like struktures. They are paired and are thought to be the uncoiled chromosomes since they are broken into fragments by treatment with DNase. The greater part of the threads carries lateral loops like the lampbrush chromosomes of Amphibia (Fig. 14). A smaller part has no loops hut bears a series of conspicious granules with bright appearance under positive phase contrast optics (pearl-string segments) (Fig. 2). — The visibility of the chromosomes has been investigated in solutions with several ions. In hypertonic media the chromosomes contract, the granules fuse, and the pearl-string segments become lumpy (Fig. 21). In nitrogenous atmosphere and if kept at low temperature the pearl-string structures are likewise transformed into a few lumps (Fig. 19). After return to normal conditions they reconstitute their characteristic beaded appearance. — In autoradiographs obtained by injection of H³-uridine into the body cavity and by incubation of isolated nuclei in vitro, a rather uniformly distributed labelling occurs over the oocyte nuclei up to 30 min incubation time (Fig. 23). With prolonged incubation the activity of the pearl-string segments becomes more intense than the labelling of the lampbrush chromosomes (Fig. 24). After treatment with actinomycin RNA synthesis is stopped, the pearl-string axes and the lampbrush chromosomes contract, and the granules disappear more and more (Figs. 25-28). — The pearlstring segments look very much like the nucleoli in the oocytes of Amphibia, where the nucleolar substance is likewise distributed as a series of beads of rather uniform size on an axis. Therefore, the pearl-string structures may have nucleolar function in Locusta too. If so, the only difference to the Amphibian nucleoli would be the continued attachment to the lampbrush chromosomes.     

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.