Abundance of protein-bound sulfhydryl and bisulfide groups at chromosomal nucleolus organizing regions

Silver stainability of the chromosomal nucleolus organizing regions that contain the structural genes for ribosomal RNA can be abolished by proteolytic and oxidative treatments. Histone extraction has no effect. This indicates that reducing groups of non-histone chromosomal proteins are responsible for silver staining. Treatment with fluorescent sulfhydryl and disulfide specific reagents followed by silver staining demonstrates coincidence of silver dots and brightly fluorescent spots at the short arms of human acrocentric chromosomes where ribosomal RNA-genes are located. After treatment with cupric sulfite reagent in the presence of urea fluorescence and silver staining was no longer possible. Silver staining has been reported to be associated with ribosomal RNA-gene activity. Acrocentric chromosomes that are negative in silver staining also lack the brightly fluorescent spots. Therefore, we conclude that an abundance of protein-bound sulfhydryl and disulfide groups occur at nucleolar organizing regions with active genes. Differentially fluorescing spots could not be observed after staining with fluorescamine. So, either the sulfhydryl reagents used in this study are much more sensitive than fluorescamine to study protein distributions in cytological preparations, or our observations point to a local accumulation of some specific protein(s) rich in sulfhydryls. The presence of many sulfhydryl and disulfide groups at the nucleolus organizing regions seems suggestive of a great flexibility of protein(s) by transition of sulfhydryl groups to disulfide bridges and vice versa at these highly active regions of the genome.     

Staining Plant Cells with Silver. III. The Mechanism

Silver does not stain all cytological structures with the same intensity. The chemical basis for differential silver staining is unclear, but differences in protein side groups available to react with silver are likely involved. These include amine, carboxyl, phosphate, sulfhydryl and hydroxyl moieties. Here we report an investigation of the chemical groups that could be involved in salt-nylon silver staining of onion root tip squashes. Based on the results, we conclude that SN silver staining primarily depends on the presence of tyrosine hydroxyl groups, and we propose a mechanism for staining.     

Identification of nucleolus organizer regions (NORs) in normal and neoplastic human cells by the silver-staining technique.

Silver nitrate has been used to demonstrate the chromosomal location of ribosomal cistrons in nine tissue-culture lines derived from human tumors of various pathological origins. Control individuals have a particular modal number (range 7--10) of D- and G-group chromosomes stained with silver. In the controls, 96.2% of the D- and G-group chromosomes that have a stalk show silver staining, while no relationship can be seen in acrocentric chromosomes without stalks. The tumor cells, whose modal chromosome numbers range from 42 to 68, possess variable numbers of acrocentrics (11--18). The number of chromosomes stained with silver, however, remained at control levels (range, 6--9). These data indicate that, in humans, silver staining may not identify all NORs that contain structural ribosomal genes.     

Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis

In proteomics it is essential to be able to detect proteins separated by gel electrophoresis at high sensitivity. Silver staining is currently the most popular method. Here we present silver staining protocols that are optimized for staining sensitivity, peptide recovery and compatibility with digestion and mass spectrometry.     

The mechanism of silver staining of proteins separated by SDS polyacrylamide gel electrophoresis

Gel based silver staining of proteins is thought to occur by selective reduction of silver ions to insoluble metallic silver at specific initiation sites in the vicinity of the protein molecules. Silver stained protein bands generally are dark brown or black with considerable variation in color intensity. The color variation has been attributed to diffractive scattering by silver grains of different sizes. Our experiments, however, demonstrate that color variation is due to the formation of silver chromate deposits that are incorporated into formalin fixed proteins. Understanding the mechanism of silver staining is essential for developing a method for protein quantification.     

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Silver staining,immunofluorescence, and immunoelectron microscopic localization of nucleolar phosphoproteins B23 and C23

Nucleolar organizer region (NOR)-specific silver staining and immunolocalization of nucleolar phosphoproteins B23 and C23 were compared in Novikoff hepatoma ascites cells. Silver staining and protein C23 immunostaining were both localized in the fibrillar shell surrounding the fibrillar center and in the fibrillar center. During mitosis, silver staining and protein C23 were localized at the NORs. Therefore, protein C23 and the silver-staining protein both seem to be associated with rDNA-containing structures (Mirre and Stahl 1981). A comparison of toluidine blue staining specific for RNA and B23 immunostaining demonstrated that protein B23 was associated with RNA-containing regions of the nucleolus and was absent from the fibrillar centers. Localization of these proteins and their functions are discussed in relation to the organization of the nucleolus.     

Further studies on the cytochemistry of the standardized silver staining of interphase nucleoli in smear preparations of Yoshida ascitic sarcoma cells in rats

Silver staining procedure for the selective demonstration of nucleolar silver stained granules (SSG) and for the simultaneous demonstration of SSG and nucleolar silver stained matrix (SSM) were studied in smears of rat Yoshida sarcoma cells. The successful results of these procedures depend mainly on the quality of silver nitrate and formaldehyde. However, both chemicals can be easily standardized and stabilized disregarding their origin and batch. In standardized procedures (one-step procedure for the selective demonstration of SSG and two-steps procedure for the simultaneous demonstration of SSG and SSM) the silver is apparently bound to acidic groups of proteins of SSG and SSM. The proteins of SSG and SSM seem to be different but both belong to the group of acidic non-histone proteins. According to the results of digestion experiments a possibility also exists that the acidic proteins of SSG may be associated with DNA. The identification of SSG visualized by described standardized procedures was determined not only by cytochemical extraction tests but also by biological experiments. The latter demonstrated that the number of SSG in Yoshida sarcoma cells decreases after treatment of experimental animals with actinomycin D and therefore depends on the state of the nucleolar RNA synthesis.     

Further studies on the cytochemistry of the standardized silver staining of interphase nucleoli in smear preparations of yoshida ascitic sarcoma cells in rats

Summary Silver staining procedure for the selective demonstration of nucleolar silver stained granules (SSG) and for the simultaneous demonstration of SSG and nucleolar silver stained matrix (SSM) were studied in smears of rat Yoshida sarcoma cells. The successful results of these procedures depend mainly on the quality of silver nitrate and formaldehyde. However, both chemicals can be easily standarized and stabilized disregarding their origin and batch. In standardized procedures (one-step procedure for the selective demonstration of SSG and two-steps procedure for the simultaneous demonstration of SSG and SSM) the silver is apparently bound to acidic groups of proteins of SSG and SSM. The proteins of SSG and SSM seem to be different but both belong to the group of acidic non-histone proteins. According to the results of digestion experiments a possibility also exists that the acidic proteins of SSG may be associated with DNA. The identification of SSG visualized by described standardized procedures was determined not only by cytochemical extraction tests but also by biological experiments. The latter demonstrated that the number of SSG in Yoshida sarcoma cells decreases after treatment of experimental animals with actinomycin D and therefore depends on the state of the nucleolar RNA synthesis.     

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei

Summary Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver.In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes.In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules.In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes.In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes.In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components.The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.     

Differences in accumulation and phosphorylation of proteins in vegetatively growing and aggregation-competent cells of Dictyostelium discoideum.

A comparison of proteins from whole cell lysates of vegetative amoebae and aggregation-competent cells by high-resolution two-dimensional gel electrophoresis coupled with a sensitive silver staining method revealed distinct differences. In aggregation-competent cells, 16 proteins present in the vegetative amoebae disappeared, and 25 new proteins appeared. A few other proteins showed quantitative variation during the transition of vegetative amoebae to aggregation competence. Identification of phosphoproteins by in vivo labeling with [32P]orthophosphate showed that none of the developmentally regulated cellular proteins were modified. Phosphorylation was observed in four proteins. One protein was phosphorylated exclusively in aggregation-competent cells. The phosphorylation level of two other proteins was higher in aggregation-competent cells compared with vegetative amoebae. The data suggest that phosphorylation of cellular and certain ribosomal proteins may be regulated coordinately in Dictyostelium discoideum.     

Karyotype analysis, banding, and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea : Scarabaeidae)

Conventional staining, differential banding, and in situ hybridization with both ribosomal and telomeric probes to mitotic chromosomes of Gymnopleurus sturmi (Scarabaeoidea : Scarabaeidae) are described. The karyotype is distinguished by a pericentric inversion polymorphism in chromosome 3, which is either acrocentric or subtelocentric. Silver staining (Ag-NOR) and chromomycin A3 (CMA3), failed to study the detection of nucleolar organizer regions (NORs), due to the extensive silver and CMA3 stainability of all GC-rich heterochromatin. Fluorescent in situ hybridization (FISH) using a Paracentrotus lividus (Echinodermata) rDNA probe mapped the ribosomal RNA genes (rDNA). FISH with the all-human telomeric sequences (TTAGGG)n revealed a lack of homology between the telomeric probe and the telomeres of G. sturmi. This suggests that the telomeric hesanucleotide (TTAGGG)n is not so conserved within eukaryotes as it has been hypothesized.     

Enhanced thiosulfate-silver staining of proteins in gels using Coomassie Blue and chromium

Potassium chromium sulfate, a new sensitivity enhancer for silver staining of proteins in gels, enhanced the sensitivity of the thiosulfate-silver staining method. The sensitivity could be further improved when potassium chromium sulfate was used in association with another sensitivity enhancer, Coomassie Brilliant Blue R-250 (CBB R-250). The sensitivity of the CBB-chromium modified method to strongly basic proteins such as ribosomal proteins was about 20-fold over that of the published method. This novel method has direct applicability for 2-D gel electrophoresis used in proteomics.     

Requirement for cysteine in the color silver staining of proteins in polyacrylamide gels

To determine whether cysteine residues have a contribution to the mechanism of color silver staining, we silver stained sodium dodecylsulfate polyacrylamide gel electrophoresis separations of proteins which have few or no cysteines. Proteins without cysteine stained negatively (yellow against a yellow background) with silver. Proteins with one or more cysteines stained orange, red, brown, or green/gray depending on the mole percentage of cysteine and whether they contained covalently attached lipids. The colors could not be correlated with the mole percentages of cysteine of these proteins indicating that some components other than cysteine affect the staining color of cysteine-containing proteins. Silver staining of amino acids, sugars, nucleotide bases, or lipopolysaccharide dot-blotted onto nitrocellulose paper implicated adenine, lipids, the basic amino acids, and glutamine, but not sugars or other amino acids in silver/protein complexes.