NOR-silver Staining Research Articles

32. Jumping genes in frogs; cytogenetic evidence that additional ribosomal gene locations in Hyla chrysoscelis (Cope's gray treefrog) are associated with mobile genetic elements

Cope's gray treefrog, Hyla chrysoscelis is composed of a complex of 5 cryptic species which occupy distinct geographical areas within the range of the currently recognized species and each species is defined by C-band patterns and the constitutional location of the 18S and 28S rRNA genes. In addition to the usual constitutional position of the ribosomal genes, there are extra sites detectable by FISH in all of the putative species of the complex. Many of the extra rRNA sites cannot be detected by NOR silver staining, indicating that they are not active, so they have to be analyzed using rRNA FISH probes. In over 200 individuals that have been analyzed so far by FISH, the location of the extra rRNA sites have been found to be unique to each individual. It has been hypothesized that mobile genetic elements of some type may be responsible for the polymorphisms found in the location of rRNA sequences in frogs and a number of other organisms, including plants. In order to test this hypothesis in gray treefrogs, a population with the constitutional rRNA gene position on the long arm of chromosome 8 (range east of the Appalachians from Virginia to Georgia) was chosen for study. Amplexed (mating) pairs were collected in the wild and placed in aquaria to complete egg laying. The parents and some of their progeny (tadpoles) were analyzed by FISH with a 28S rRNA probe. FISH analysis revealed that all of the tadpoles had the same constitutional rRNA gene locations as the parents, but they differed from their parents and each other in the location of the extra rRNA gene sites. This evidence supports the hypothesis that mobile elements may have an association with extra rRNA sites in H.chrysoscelis.

Cytogenetic Studies and Correlate Considerations on Rhamdiinae Relationships (Pisces, Siluroidei, Pimelodidae)

In Pimelodidae fish the available chromosome numbers range from 2n=46 to 2n=63, the value 2n=56 being the most frequent one. In the present paper 6 species belonging to the Rhamdiinae subfamily were analyzed: Rhamdia hilarii, R. quelen, Cetopsorhamdia sp., C. iheringhi, Imparfinis cf. piperatus and Imparfinis aff. schubarti. Chromosome preparations were obtained by direct and short term culture methods from kidney cells and analysed under standard Giemsa staining, C-banding and NOR silver staining. Rhamdia species present a basic karyotype composed by 58 chromosomes and the NORs are located on the short arm of a subtelocentric chromosome pair. Cetopsorhamdia sp., C. iheringhi and Imparfinis aff. schubarti. also shows 2n=58, while I. cf. piperatus is the only analyzed species presenting 2n=56. However, these 4 species show an interstitial NOR location. These chromosomal data agree with the occurrence of subgroups in the Rhamdiinae subfamily, proposed on the basis of their morphological traits.

HeLa D98/AH-2 studied by chromosome painting and conventional cytogenetical techniques

Metaphase chromosomes of HeLa D98/AH-2 cells were studied by GTG-banding, NOR-silver staining and in situ hybridization using libraries specific for each human chromosome as probes (chromosome painting). The structure and composition of all marker chromosomes could be determined, allowing a critical assessment of earlier studies. The revised HeLa D98/AH-2 karyotype should provide a reference standard in certain cancer cytogenetic studies.

Cytochemical variations in the nucleolus during spermiogenesis in man and monkey

The fine structure, nature and fate of the components of the nucleolus were studied in young (steps 1, 2), intermediate (steps 3, 4, 5) and mature spermatids (steps 6, 7, 8) of man and monkey, by use of several cytochemical techniques (alcoholic PTA; sodium tungstate: EDTA; HAPTA; nuclease-gold complexes; NOR silver staining). As controls, comparative ultrastructural and cytochemical observations of the nucleolus in spermatids and Sertoli cells were made in the same sections of seminiferous tubules. In the young spermatids of the two species studied, the nucleolar masses exhibited identical features. Segregation of the nucleolar components took place in the nuclei of step 1 spermatids. No typical fibrillar center was observed. In spermatids at steps 1 and 2, the nucleolar masses appeared to be made up of two fibrillar components of equal density, one spherule-shaped, the other forming cords, both surrounded by clusters of 15-20 nm-diameter granules. Alcoholic PTA and sodium tungstate yielded a selective positive contrast of the two fibrillar components whereas EDTA and RNase-gold reacted with the peripheral granular material. Treatment with RNase-gold and DNase-gold complexes resulted in preferential labeling at the periphery of the fibrillar components. After NOR silver staining, numerous small silver grains were localized over the fibrillar cords, suggesting the persistence of specific acidic non-histone proteins. On the contrary, the spherule was never stained. In intermediate spermatids, when the nucleolar components were dissociated, scattered clusters of granules stained by EDTA and HAPTA remained in the entire nucleoplasm. Nucleolar disintegration was accompanied by dispersion of argyrophilic material.(ABSTRACT TRUNCATED AT 250 WORDS)

Ag-NOR proteins and rDNA transcriptional activity in plant cells.

In this work we used Allium cepa root meristem cells in actively growing conditions and under treatment with the protein synthesis inhibitors cycloheximide (CHM) and puromycin. Morphological and quantitative results indicate that these drugs induce dramatic alterations in nucleolar structure reflected by a decrease of nucleolar size, much more evident under treatment with CHM, and by segregation of its main components. Quantitative analysis shows a decrease in NOR-silver staining after treatment with CHM, whereas in cells treated with puromycin NOR-silver staining remains constant. Our results reveal a decrease in the Ag-NOR proteins under conditions of diminished cell activity, suggesting a direct relationship between the quantity of Ag-NOR proteins and transcriptional activity. Using two-dimensional gel electrophoresis and NOR-silver staining in gels, we have characterized some proteins corresponding to molecular weights of 28 and 31 KD and pI of approximately 5.2. After treatment with CHM, reactivity of these proteins against NOR-silver staining is diminished. By means of a morphological study, analysis of NOR-silver staining, and of anti-DNA and RNAse-gold labeling, we have tried an approach to the nucleolar organization in plant cells. Our results suggest that the fibrillar component shows a reticular distribution where fibrillar centers, as described in animal cells, are not distinguished.

An experimental approach to nucleolar organization in plant cells: A morphological, cytochemical and quantitative study

ABSTRACT We have used morphological, cytochemical and immunocytochemical techniques, along with transcription inhibitors, to try to understand the nucleolar organization in Allium cepa root meristem cells. Our results reveal that the fibrillar component shows intense staining after using the NOR-silver technique, and gold particle labelling after incubation with DNase-gold and RNase-gold complexes. These different techniques reveal a reticular structure of the fibrillar component, supported by a central axis that is circular in transverse section. These structures are usually considered to be fibrillar centres, but we have called them clear areas, since we find in their interior neither Ag-NOR proteins nor labelling after incubation with nuclease-gold or anti-DNA. Taking into account the specific localization of the Ag-NOR proteins, our cytochemical and quantitative results, after applying NOR-silver staining in nucleoli of control cells and cells treated with transcription inhibitors, help us to a better understanding of the mechanisms of action of these drugs. Our results support a structural and functional role for the Ag-NOR proteins throughout the course of transcription.

Intracellular localization of argyrophilic proteins in the maturing oocyte, fertilized egg, and spermatozoa of the starfish, Asterina pectinifera

Changes in intracellular localization of argyrophilic proteins visualized as silver-stained particles by nuclear organizer region (NOR)-silver staining were investigated in starfish oocyte maturation. The silver-stained particles were localized in the germinal vesicle and nucleolus of immature oocytes and dispersed into the cytoplasm at the time of germinal vesicle breakdown (GVBD). In the mature egg cytoplasm, silver-stained particles were distributed on yolk-like granules with diameters of 0.3-1.0 microns. In spermatozoa, silver-stained particles were detected heavily in the acrosome and centrosomes but few were detected in the nucleus, whereas they were present in the male pronucleus of fertilized eggs. The silver-stained particles were removed by pretreatment of eggs with protease but not with nuclease. These results indicate that argyrophilic proteins disperse to the egg cytoplasm during GVBD and might be incorporated to the male pronucleus from the egg cytoplasm in fertilization. The morphological changes from chromosomes through chromosome vesicles to female pronucleus were also observed with light microscopy after NOR-silver staining.

Further evidence that phosphoprotein C23 (110 kD/pI 5.1) is the nucleolar silver staining protein

Previously we demonstrated a similar distribution between nucleolar organizing region-(NOR)-specific silver staining and localization of nucleolar phosphoprotein C23 (MW 110 kD/pI 5.1) [1, 2]. We now report that under Fixation conditions which allow for antibody binding and subsequent silver staining, monoclonal antibody against protein C23 blocks NOR silver staining as well as silver staining in interphase nucleoli. Monoclonal antibody against nucleolar phosphoprotein B23 (MW 37 kD/pI 5.1) did not block silver staining in either NORs or interphase nucleoli. These, along with earlier observations, provide evidence that nucleolar phosphoprotein C23 is the major silver staining protein of the nucleolus and that it is directly or indirectly associated with rDNA.

Visualization of Ag-NOR proteins on nucleolar transcriptional units in molecular spreads.

Application of NOR silver staining to Pleurodeles oocyte nuclei showed selective silver deposits on the fibrillar part of the nucleoli in situ. This staining was adapted to nucleoli spread on grids, by a procedure which allowed the spreading of transcription units from both nucleoli and lampbrush chromosomes on the same grids. This permitted localization of the predominant nucleolar Ag-stained proteins on the nucleolar transcriptional units and not on the lampbrush chromosomes. These proteins were found exclusively on the transcribed part of the nucleolar genes and were not seen in apparently untranscribed spacer regions. The proteins seemed preferentially located on the DNP axis rather than on the RNP fibrils.

The morphological relationship in electron microscopy between NOR-silver proteins and intranucleolar chromatin.

The relative distribution of NOR proteins and chromatin fibers in the nucleoli was visualized in human cell line. The chromatin was revealed by a Feulgen-like procedure using osmium-ammine as DNA tracer. This selective staining was combined with NOR-silver staining. We provide morphological evidence for constant overlapping of the silver deposit sites with dispersed intranucleolar chromatin fibers. Silver stained proteins were sometimes observed in contact with the chromatin fibers, suggesting that at least some of the Ag-NOR proteins might be closely connected with the dispersed nucleolar DNA.

Sex vesicle-associated nucleolar organizers in mouse spermatocytes: localization, structure, and function

Selective silver staining demonstrated that autosomal bivalents containing transcriptively active nucleolar organizers associated with the sex vesicle during pachytene of mouse spermatocytes. Later in pachytene, the nucleolar organizers covered the portion of the sex vesicle furthest from the attachment to the nuclear envelope. Hybridization in situ revealed the presence of rDNA in the silver-positive material. The nucleolus, formed from an autosomal bivalent, exhibited a large fibrillar center surrounded by an electron-opaque fibrillar zone. The nucleolar association with the sex vesicle was studied at early, middle, and late pachytene by hybridization in situ, NOR silver staining, and electron microscopy. These observations enabled us to further define the relationships of the nucleolar components with the X-Y pair.