The nucleolus organizers of Plethodon and Aneides located by in situ nucleic acid hybridization with Xenopus 3H-ribosomal RNA

The main clusters of ribosomal genes, or nucleolus organizers, have been located by in situ nucleic acid hybridization of Xenopus laevis ³H labelled ribosomal RNA to mitotic chromosomes in squash preparations of intestinal epithelium from 7 species of Plethodon and 3 species of Aneides. The species used were chosen on account of having well known karyotypes and genome sizes. The Plethodon species covered a range of genome size of 20–69.4 pg. The locations of those nucleolus organizers that could be detected by autoradiography after in situ hybridization varied from species to species, and in Aneides there were differences between two populations of the same species. On the other hand, some distantly related species of Plethodon, with widely different genome sizes, had nucleolus organizers at corresponding positions. The results are discussed in relation to ideas on karyotype stability, homosequentiality and chromosome repatterning.     

Differences in the number of nucleolus organizers in Chironomus tepperi shown by in situ hybridization

Two stocks of Chironomus tepperi could be isolated. One stock, N(IV)⁺, contains nucleolus organizers in chromosome I and IV, whereas the other one, N(IV)^–, shows only one nucleolus in chromosome I. It is demonstrated by in situ hybridization with radioactive rRNA that the absence of the nucleolus in chromosome IV of stock N(IV)^– is not related to an inactivation of the nucleolar DNA, as might have been suggested, but is due to the lack of ribosomal cistrons.     

Silver staining of the lampbrush chromosomes ofTriturus cristatus carnifex

Lampbrush chromosomes fromTriturus cristatus carnifex were stained using the ammoniacal silver staining (AgAS) technique. Many of the recognized marker structures proved to be silver positive, plus between six and fifteen lateral loop pairs. None of the stained loop pairs corresponded to known sites of the nucleolus organizers, although the extrachromosomal nucleoli were silver positive. The ammoniacal silver staining technique does not demonstrate the specificity for active ribosomal cistrons in lampbrush chromosomes that it does in a wide variety of mammalian mitotic chromosomes.     

Localization of ribosomal cistrons at the ultrastructural level by in situ hybridization technique

In situ hybridization with 3H 28 S ribosomal RNA at the ultrastructural level shows labelling exclusively over the nucleolus and specifically over the dense nucleolar component (DNC). These findings clearly reveal that the ribosomal cistrons are located in the DNC of the nucleolus.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7     

Individual quantitative rDNA variation in three species of the Cucurbitaceae family

Individual quantitative variation in rDNA content within three species of the Cucurbitaceae family has been studied by rRNA/DNA filter hybridization experiments. The results showed a 2.3-fold range of variation in the number of ribosomal cistrons per diploid cell in an Ecballium elaterium natural population. This range of variation is compared with the smaller range observed in three Cucumis sativus and in two Cucurbita pepo varieties obtained as F₁ hybrids between pure lines.This work was supported by CNR Contract No. 74/0267.     

Detection of rRNA and phaseolin genes on polytene chromosomes of Phaseolus coccineus by fluorescence in situ hybridization after pepsin pretreatment.

This is the first report of fluorescence in situ hybridization (FISH) on plant polytene chromosomes. Different protease pretreatments have been tested to improve fluorescence in situ hybridization FISH on polytene chromosomes of a plant, Phaseolus coccineus, with the aim to enable the detection of low-copy genes. The structural preservation of the chromosomes and the distinctness of the FISH signals were comparatively analysed with a probe for the ribosomal RNA genes after digestion with pepsin and trypsin. The pepsin pretreatment resulted in a general loosening of chromatin with good conservation of chromosome morphology and an increased number and density of signal points. The six nucleolus organizers exhibited significant differences in condensation. The pretreatment with pepsin enabled the detection of the low-copy genes encoding the seed storage protein phaseolin.     

Chromosomal location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

The 5S ribosomal RNA genes have been localized in mitotic and lampbrush chromosomes of Triturus vulgaris meridionalis by in situ hybridization. These genes are clustered in a single locus in an intercalary position of the long arm of chromosome XI. In lampbrush chromosome XI the 5S genes are located near a loop landmark mapped at 66 units.     

Cytogenetics of the parthenogenetic grasshopper Warramaba virgo and its bisexual relatives

A study of the chromosomal location and genomic organization of the ribosomal RNA cistrons in the genus Warramaba, involving in situ hybridization and restriction enzyme analysis as well as C- and N-banding and silver staining, has confirmed that the parthenogenetic species W. virgo has two phylads. These phylads appear to have originated independently by hybridization between the precursors of the present day bisexual species P169 and P196. The clones of the Standard phylad of W. virgo have their 18S+26S rDNA cistrons located in C-bands 4, 44 and 49, while those of the Boulder-Zanthus phylad have them in C-bands 50, 74 and 87.5. The relative numbers of the ribosomal genes at the different sites vary greatly from clone to clone and are closely correlated with the width of the corresponding C- and N-bands. Site 49 of the ribosomal cistrons is present as a separate band in the eastern race A of P196 but has been incorporated into band 50 in the western race B of this species. The former race is assumed to be ancestral to the Standard phylad of W. virgo, the latter to the Boulder-Zanthus phylad, but there has been loss of the 74 and 87.5 sites in the the Standard phylad and the 4 and 44 sites in the Boulder-Zanthus clones. The ribosomal cistrons in W. picta, a species with a primitive karyotype, occur in several sites, only some of which have counterparts in P169 and P196. The 5S rDNA cistrons are located in bands 59.5, 69 and 72.5 in the Standard phylad of W. virgo. — The genomic organization of the 18S+26S rDNA cistrons, as shown by restriction enzyme analysis, is different in the two W. virgo phylads and there are also differences in organization between P196A and P196B. The pattern in P196B and that in the Boulder-Zanthus phylad suggest that they are related. As in the in situ analyses, the genomic organizations of the ribosomal cistrons in both W. virgo phylads are not simply the additive products of those in any known populations of P169 and P196. New repeat lengths indicative of segmental amplification events occur in particular clones of W. virgo. — Throughout the genus Warramaba the N-banding technique stains all bands containing 18S+26S and 5S rDNA cistrons. The Olert silver technique stains band 72.5 in the Standard phylad, but does not correlate with the locations of 18S+26S ribosomal genes.     

Functional features of the nucleolar organizer in developing oocytes of juvenile birds

The genes of rRNA in the nucleolar organizer region (NOR) are inactivated in the oocytes of adult birds despite the functioning of lampbrush chromosomes. The nucleolus is not formed during all stages of the oocyte development. On the other hand, two morphological forms of oocytes differing by the presence of nucleolus in the germinal vesicle are described in the ovaries of juvenile birds. The activation and function of the ribosomal genes in avian oogenesis is still vague. In this work, the NOR activation in chicken (Gallus gallus domesticus) oocytes is confirmed with the help of fluorescence immunohistochemistry (antibodies against nucleophosmin, fibrillarin, and UBF1) and in situ nucleic acid hybridization (FISH with the probe to ITS1 in pre-rRNA). It is demonstrated that the nucleolus in the oocytes at the lampbrush stage in the chicken ovaries is fragmented after complete inactivation of the ribosome genes: the nucleolar fragments contain fibrillarin but do not contain pre-rRNA molecule. The utility of the ovary 3D reconstruction using serial histological sections for quantification of sex cell population heterogeneity in the ovaries of juvenile birds is demonstrated. The obtained results improve the current insight into the functional NOR state in the oocytes of juvenile female birds and contribute to the concept of diversity in the scenarios of gametogenesis.     

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.     

Further Studies on the Ribosomal RNA Cistrons of SCIARA COPROPHILA (Diptera)

Additional experiments with homologous as well as heterologous hybridization confirmed our previous finding in Sciara coprophila that XX females have nearly twice the number of ribosomal RNA cistrons as XO males. A comparison between two different X' chromosomes revealed that only the one carrying the irradiation-induced Wavy mutation has a deletion of 70% of its ribosomal RNA cistrons as compared to the standard X. The deletion is relatively stable, and the remaining ribosomal RNA cistrons donot appear to undergo disproportionate replication or magnification as in Drosophila. Homologous hybridization experiments revealed an unusually low reiteration of ribosomal RNA cistrons in this fly, 45 gene copies per X chromosome. The question is raised as to whether such a low number of cistrons may be related to the unusual nucleolar condition encountered in the Sciaridae.     

Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining

A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.