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.     

An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes

Summary A reliable technique for staining human chromosomal nucleolar organizers (NOR's) with silver solutions is described. The NOR's can be selectively stained dark brown by silver solutions leaving the chromosome arms unstained and available for counterstaining with orcein or Giemsa dyes. Unequivocal identification of chromosome pairs bearing NOR's can be achieved using fluorescent banding techniques followed by silver staining. The silver staining procedure for NOR's was simplified and standardized through control of the chemical and physical conditions during silver impregnation and developing.     

Preferential association of nucleolar organizing human chromosomes as revealed by silver staining technique at mitosis

Summary The frequency of different types of satellite associations of nucleolar organizing human chromosomes (i.e. acrocentric chromosomes; 13, 14, 15, 21, and 22) is reported using 10 normal individuals by Ag-staining technique. The preferential involvement of acrocentric chromosomes in satellite association is suggested. Only acrocentric chromosomes with active NORs (i.e. Ag-stained) were found in association while unstained (inactive NORs) chromosomes were never seen in satellite association. In general as number of NORs expression increase, the frequency of association per cell was also increased. A possible mechanism and the clinical consequences of such an unusual phenophenon is described.     

Nuclei and nucleolar organizing regions in chromosomes of lymphocytes on different stages of periodic disease

By scanning cytomorphometry a cytological study was first performed on the behavior of nuclei and nucleolar organizing regions (NOR) in chromosomes of peripheral blood lymphocytes of healthy men and of patients with periodic disease (familial Mediterranean fever, FMF) on different stages of development, including its complication with amyloidosis. The volume and total surface of nuclei, the sum total volume and sum total surface of NOR, the mean number of NOR for one nucleus and distribution of nuclei according to NOR number were measured. It is shown that the parameters of nuclei and NOR for patients with FMF on all stages clearly and trustworthy differ from those for healthy men. They are sufficiently informative, can be successfully used in clinical practice and even serve as an early diagnostic test for amyloidosis complication.     

Nucleolar organizing regions of human chromosomes

Summary Silver-Stained cells from 49 parents with a history of several abortions were compared with cells from 35 parents with normal liveborn children. The modal and mean number of silver-stained NORs (Ag-NORs) observed on D- or G-group chromosomes was similar in both groups and between males and females. Ag-NORs were randomly distributed on all five acrocentric pairs. The distribution and size of Ag-NORs within an individual was not random and was fairly consistent from cell to cell.The mean number of associations per cell was similar in both males and females of the abortion group and was less than the number of associations in controls. The probability of D- or G-group chromosomes being associated was near the expected probability of 0.6 for D-association and 0.4 for G-association. The frequency of association of any chromosome combination did not differ statistically from the expected values, though the number of associations, 15/22, was higher than expected.     

Combined silver staining of the nucleolus organizing regions and Giemsa banding in human chromosomes

Summary A combination of the silver-staining method of the nucleolus organizer regions (NORs) with a Giemsa-banding method is deccribed. This double staining allows a rapid identification of the NOR-bearing chromosomes.Supported by the Deutsche Forschungsgemeinschaft (Za 32/14).     

Silver staining of the nucleolar organizer regions (NORs) in semithin Lowicryl sections

The one-step silver technique was applied to semithin Lowicryl sections of root meristem cells of Allium cepa and a human tumor cell line (TG cells). In vegetal cells, after 5 min of staining reaction, the Ag-NOR proteins formed ring-shaped structures peripherally within the nucleolus. In animal cells silver granules were distributed over the entire nucleolus. The specificity of the staining reaction was increased by incubation of the sections in NH4Cl and Schiff's reagent prior to Ag-NOR silver staining.     

Silver staining of histone-depleted metaphase chromosomes

To investigate a possible relationship between the core-like structures seen in silver-stained chromosomes (prepared by standard cytogenetic methods) and the scaffolds observed in histone-depleted chromosomes, the ability of the scaffold to stain with silver has been examined. Isolated chromosomes were histone-depleted by washing in ammonium acetate or by spreading the chromosomes on an ammonium acetate hypophase. The residual chromosome structures were carbon-platinum shadowed or stained with silver, and then examined by electron microscopy. The results provide clear evidence that the scaffold structure has a high affinity for silver and is therefore similar in its silver-staining potential to the core structure in standard chromosomes. This suggests that the silver core in standard chromosomes may represent the scaffold visualized by histone depletion. The peripherally dispersed DNA radiating from the scaffold also proved to be silver-reactive, and additional experiments demonstrated that purified DNA is capable of binding silver. This result indicates that cytological silver staining is not simply a matter of staining protein, as has previously been thought, but may also involve the staining of chromosomal DNA. In the ammonium acetate-treated and carbon-platinum-shadowed preparations, the scaffold structure was highly variable in its morphology and appeared to be composed of undispersed or incompletely dehistonized chromatin fibers. The silver-stained scaffold reflected this variability. Taken together with other evidence, these findings lead to a questioning of the reality of chromosome core structures.     

Silver staining of the nucleolar organizer regions (NORs) on Lowicryl and cryo-ultrathin sections

Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.     

Structural organization of ribosomal cistrons in human nucleolar organizing chromosomes

The variable morphology of nucleolar organizer regions (NORs) of human acrocentric chromosome 13 was studied in detail by the N-banding technique. It was demonstrated that NOR may become further segmented and several hypotheses have been proposed for structural variability. The polymorphic nature of NORs may have an important significance in human evolution.     

Mitotic association patterns of nucleolar organizing chromosomes in the mouse.

Following the report of localization of nucleolar organizer regions to chromosome pairs 15, 18, and 19 of the mouse, a study was undertaken to test these chromosomes for association during mitotic metaphase. Cells from sevel strains of mice were analyzed, and no strong evidence was found for any significant association patterns.     

A technique for the simultaneous staining of both nucleolar organizer regions and kinetochores of human chromosomes with silver.

Pretreatment of human metaphase chromosomes with NaOH at a pH of 8.5, followed by staining with silver nitrate, differentially stains both the nucleolar organizer regions on the 10 acrocentric chromosomes as well as the kinetochore centers on all 46 chromosomes.     

Analysis of nucleolar organizing regions in parents of trisomic spontaneous abortions

Summary Nucleolar organizing region (NOR) variants of parents of karyotyped spontaneous abortions were examined to fest the hypothesis that double NORs are important in the genesis of acrocentric trisomies. We were unable to detect any significant difference in the frequency or types of NOR variants between parents of acrocentric trisomies and parents of other types of spontaneous abortions, nor did we identify a double NOR in either group. Thus, it seems unlikely that double NORs are of major significance in the etiology of acrocentric trisomies.     

Decreased number of nucleolar organizing regions in collagen lattice cultured fibroblasts

Dermal fibroblasts cultivated in tridimensional matrices (lattices) of collagen exhibit a very low metabolic activity, and a low protein synthesis in particular. We have previously shown that ribosomal RNA content and half-life were decreased in collagen lattice cultured fibroblasts when compared to monolayer cultured fibroblasts. In this study, we seeded fibroblasts in collagen lattices and investigated the influence of matrix on the number of nucleolar organizing regions. We found that fibroblasts in fully retracted lattices exhibited a significant decrease of 45 % (P < 0.001) in the number of nucleolar organizing regions when compared to monolayer cultured fibroblasts. This decrease was correlated to the decrease in ribosomal RNA content. These data suggest that extracellular matrix induces early alterations of synthesis and/or processing of ribosomal RNAs, explaining, at least partly, the resulting low metabolic activity.     

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.     

Polymorphism of nucleolar organizer regions of chromosomes in human embryos

NOR activity in metaphase chromosomes from extraembryonic and embryonic tissues of 9-12 week human fetuses was studied after standard silver staining. Significant interidividual variations in the average cummulative NOR activity was assessed by means of one-factor dispersion analysis. No significant intertissue fluctuation of NOR activity was found. Total number of NOR+ chromosomes demonstrated no correlation with the embryonic age. Steady growth of an average cummulative NOR activity respective of progressive embryonic age was proven by correlation analysis method. Unequal participation of NOR-bearing chromosomes of D- and G-groups during early embryonic development in human was shown.     

Silver staining of phosphoserine and phosphothreonine in nucleolar and other phosphoproteins

The present study was designed to determine the amino acids responsible for silver staining of nucleolar and other phosphoproteins on acetic acid urea gels. With the phosphoproteins, α -casein and phosvitin, the same golden-brown to yellow color was produced that was previously found with the nucleolar organizer region (NOR) specific proteins, B23 and C23, of Novikoff hepatoma cells (Lischwe et al., 1979). Phosphoserine- and phosphothreonine-bound glycogen polymers stained golden-brown. Dephosphorylation of α -casein by calf intestine alkaline phosphatase blocked the reaction almost completely. Proteins which were insensitive to the staining reaction were stained golden-brown by binding to CNBr-activated α -D,L-glycerophosphate.     

A highly reproducible method of nucleolus organizing regions staining in plants

A method for the specific detection of the nucleolus organizing regions (NORs) of plant chromosomes has been developed employing enzymatic maceration and successive flame-drying for chromosome spreading and incubation with aqueous 50% AgNO3 at 55-60 C. When this method was applied to metaphase chromosomes the NORs were specifically discriminated as heavily stained segments in all the plant species examined. In the satisfactory results obtained by monitoring the reaction under a microscope during the course of the silver treatment, the chromosome arms were stained yellow to light brown while the NORs were dark brown to black. The present method has the advantage of yielding highly reproducible results for the specific detection of the NORs in plant materials.