Dimorphic nucleolar organizer regions in the frog Rana blairi.

The nucleolar organizer-specific staining procedure, ammoniacal silver (Ag-AS), has been used to study the distribution and size of the nucleolar organizer regions (NORs) in chromosomes of the frog Rana blairi (Mecham, Littlejohn, Oldham, Brown and Brown). The somatic metaphase karyotype of this frog is similar to that of other frogs of the Rana pipiens species complex, numerically (2n=26) and morphologically. Secondary constrictions are detectable in untreated Giemsa-stained metaphase preparations as achromatic gaps in the long arms of a pair of submetacentric chromosomes (no. 10). These constrictions are the only regions which are deeply stained with the Ag-AS method and are thus identified as the nucleolar organizer regions (Ag-NORs). In each of the three individuals, the Ag-NORs as visualized on the homologues are of unequal length.     

正常造血细胞的核仁组成区计数定量研究

本文应用银染技术对正常造血细胞的核仁组成区进行了计数定量研究。结果显示,在粒系和红系中,随细胞的成熟,细胞中簇状ＡｇＮＯＲ的数量逐渐减少．而点状ＡｇＮＯＲ数量逐渐增多,无分裂能力的成熟细胞仅有少数点状ＡｇＮＯＲ。淋巴细胞中为一完全集结的银染颗粒,而巨核细胞内为各自分离的点状银染颗粒。本结果为正常造血细胞的核仁组成区提供了基础数值。     

Argyrophilic nucleolar organizer regions (AgNORs) in interphases and metaphases of normal and neoplastic gill cells of Macoma balthica (Bivalvia: Tellinidae) from the Gulf of Gdansk, Baltic Sea

Chromosome analysis of gill cells of different populations of Macoma balthica (L.) from the Bay of Gdansk (Baltic Sea) revealed 2 clam categories, 1 with neoplastic features and 1 without. Silver-staining was performed on interphase and metaphase cells of both categories. The mean argyrophilic nucleolar organizer region (AgNOR) count per abnormal interphase cell was significantly higher than in normal interphase cells. Normal silver-stained metaphases had 3 nucleolar organizer region (NOR) chromosome phenotypes. The location of the NORs in the most frequent phenotype (55.6% in 54 metaphases scored) was interstitial on the largest metacentric chromosome pair, Pair No. 1. Abnormal silver-stained metaphases had a higher number of active NOR sites. Different phenotypes were observed (frequency greater than 10% for 67 metaphases scored); 2 were similar to those in normal metaphases and 5 were ectopic. The higher activity of AgNORs observed in abnormal cells confirmed the diagnosis of malignant neoplasia.     

Expression of Ribosomal Cistrons of Human Chromosomes at High Resolution

The expression of ribosomal cistrons in the nucleolar organizer regions (NORs) has been studied with high resolution banding in the acrocentric chromosomes of 10 normal individuals. It was found that if a particular chromosome did not stain with silver nitrate at metaphase, then it did not stain at prophase either. Therefore, it is concluded that some of the acrocentric chromosomes have variable expression of NORs.     

Expression of ribosomal cistrons of human chromosomes at high resolution

The expression of ribosomal cistrons in the nucleolar organizer regions (NORs) has been studied with high resolution banding in the acrocentric chromosomes of 10 normal individuals. It was found that if a particular chromosome did not stain with silver nitrate at metaphase, then it did not stain at prophase either. Therefore, it is concluded that some of the acrocentric chromosomes have variable expression of NORs.     

Nucleolar organizer regions in rat urinary bladder tumors induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

The number of nucleolar organizer regions (NORs) stained by the one-step silver colloid method was measured in preneoplastic and neoplastic bladder lesions induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in rats. Male ACI/N rats, 6 weeks of age, were given 0.05% BBN in drinking water for 5, 8, 12, 18 and 30 weeks to induce preneoplastic and neoplastic transitional cell lesions. The mean numbers of silver-stained NORs (AgNORs) in such lesions were as follows: untreated transitional epithelium (n = 6), 1.26 +/- 0.09; transitional cell epithelium outside focal lesions (n = 10), 1.75 +/- 0.10; simple hyperplasia (n = 10), 2.01 +/- 0.15; papillary or nodular (PN) hyperplasia (n = 10), 2.15 +/- 0.19; transitional cell papilloma (n = 5), 2.37 +/- 0.12; transitional cell carcinoma (n = 5), 3.52 +/- 0.23. Thus, the mean number of AgNORs showed a step-wise increase from untreated and treated, histologically normal transitional epithelium through simple hyperplasia and PN hyperplasia to transitional cell papilloma and carcinoma. These results suggest that the mean number of AgNORs may reflect the proliferative nature of bladder lesions induced by BBN, as reported in preneoplastic and neoplastic lesions in other organs. PN hyperplasias were classified into two types based upon the mean number of AgNORs, indicating that they include reversible and irreversible changes in contrast with simple hyperplasia which is reversible change.     

Nucleolar dominance does not occur in root tip cells of allotetraploid Brassica species.

Using in situ hybridization and silver staining methods, the numbers of active and inactive rDNA loci have been established for three allotetraploid species of Brassica (B. napus, B. carinata, and B. juncea) and their diploid ancestors (B. campestris, B. nigra, and B. oleracea). The allotetraploid species have chromosome numbers equal to the sum of the numbers in their diploid relatives, but have fewer rDNA loci. All species investigated have lower numbers of active NORs (AgNORs, nucleolar organizer regions) compared with the numbers of rDNA sites revealed by in situ hybridization. The number of active rDNA loci of the allotetraploid species is equal to the number of AgNORs in their diploid ancestors, indicating the absence of nucleolar dominance in amphidiploid Brassica species, at least in root meristematic cells.     

The three-dimensional ultrastructure of interphasic and metaphasic nucleolar argyrophilic components studied with high-voltage electron microscopy in thick sections

The three-dimensional structure of the nucleolar argyrophilic components was studied by recording stereo-pairs of tilted thick sections--0.5-2 microns thick--observed with 200 and 300 kV high-voltage electron microscopy (HVEM). Using a very specific silver staining method, the argyrophilic components were stained with a high contrast relatively to the unstained background, thus allowing their study with a high resolution within thick sections. This study was performed on compact nucleoli (of HL60 and K562 cells), on reticulated nucleoli (of human breast cancerous cells) and on metaphasic nucleolar organizer regions (NORs). In compact nucleoli argyrophilic components show a 'knotted rope-like' structure in which knots are constituted of one central fibrillar centre surrounded at some distance by loops of the dense fibrillar component and in which the rope is constituted of dense fibrillar component. In reticulated nucleoli silver deposits are confined to the surface of the nucleolonema as several strands twisted at the periphery of the fibrillar component. During metaphase some NORs get a characteristic crescent-shaped structure disposed at the periphery of some chromosomes.     

肿瘤核仁形成区形态定量诊断的误差及规范化分析

本文采用包括自动图象分析技术在内的ＡｇＮＯＲ形态定量学方法,以大肠肿瘤为模型,进行了ＡｇＮＯＲ定量形态学研究的误差分析,以探讨肿瘤ＡｇＮＯＲ定量诊断规范化的可能。结果表明,染色条件、视场目标选择和细胞计数量是引起ＡｇＮＯＲ定量诊断的主要误差;恒定染色环境,正确选择欲测细胞及测定足够量的细胞是使ＡｇＮＯＲ形态定量诊断规范化的途径。     

Nucleolar organizer activity in wheat,rye and derivatives analyzed by a silver-staining procedure

Nucleolar activity was analyzed in wheat (Triticum sp.), rye (Secale cereale) and several types of wheat-rye derivatives using a modified, highly reproducible, silver staining procedure (Lacadena et al. 1984). A comparative analysis of the nucleolar organizer regions (NORs) of somatic metaphase chromosomes was made by phase contrast, C-banding, and silver staining. The frequency distribution of the number of nucleoli visualized at interphase by silver staining was also used to infer the activity of NORs. The results agree quite well with data from in situ hybridization reported by other authors. The behavior of euploid, ditelosomic and nulli-tetrasomic plants of common wheat showed the relative nucleolar activity of the four organizer chromosomes to be: 6B > 1B > 5D > 1A. — Several types of wheat-rye derivatives were analyzed: interspecific hybrid, triticale, addition and substitution lines, and plants with the genome constitutions, AABBDR, ABDR + 5D, ABRR, and ABRRR. In all cases the nucleolar organizer chromosome 1R of rye was suppressed by the presence of wheat chromosomes.     

Nucleolar assembly of the rRNA processing machinery in living cells

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.     

Silver staining of human acrocentrics in metaphase does not reflect an induced inhibition in NOR activity

Cytological staining with silver nitrate was used in order to study the activity of the nucleolar organizer regions (NORs) in metaphase figures from human lymphocytes exposed to mercury chloride and actinomycin D. The cells were exposed to both compounds either during G1-early S phase, allowing recovery after the exposure, or from G1 until harvest; no recovery was thus allowed in the latter case. HgCl₂ as well as actinomycin D did not influence the silver staining of the acrocentric chromosomes on metaphases. As actinomycin D is known to be an inhibitor of rRNA, as for example confirmed by inhibition of silver staining on interphase cells, our results on metaphase chromosomes indicate that AgNO₃ precipitation, although being a good indicator for nucleolar activation, is not adequate in case of inactivation.     

Metaphase association of mammalian chromosomes

The association behavior of chromosomes bearing nucleolar organizer region (NOR) and (or) C-heterochromatin in metaphase plates was analyzed. Different species with an informative chromosomal localization of NOR and C-heterochromatin were evaluated. Several examples indicate that the well-known metaphase association is not due to NORs or NOR activity per se. Other mechanisms such as ectopic pairing are responsible for the association. These types of pairing seem to be enhanced by the chromatin-decondensing effect of nearby NOR activity.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

Prognostic value of nucleolar organizer regions in neuroendocrine tumours of the lung

The value of the number and size of the nucleolar organizer regions (NORs) as prognostic indicators in human neuroendocrine lung tumours was evaluated in a quantitative study of 57 cases, including 33 small cell carcinomas (SCLCs), 9 well-differentiated neuroendocrine carcinomas (WDNECs) and 15 classic carcinoids. NORs were visualized on paraffin sections by an argyrophilic technique (AgNOR) and measured by automatic image analysis. In each case, the mean number and area of AgNORs were evaluated; the results were compared with clinical follow-up and survival. AgNOR values for both number and area were significantly higher in SCLCs than in WDNECs and carcinoids. WDNECs had insignificantly higher AgNOR values than carcinoids. Among SCLCs, AgNOR values of the oat cell subtype and the intermediate cell subtype did not differ significantly. Regardless of the histological tumour type, AgNOR values strongly correlated with prognosis, with more and larger AgNORs indicating a more progressive clinical course. In the present study we demonstrate for the first time that the biological behaviour of neuroendocrine lung tumours is correlated with the number and size of AgNORs. Thus the measurement of AgNORs may serve as an additional prognostic indicator in these neoplasms, particularly in the separation of SCLCs from WDNECs with a more favourable prognosis.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

Gold Toning Improves the Visualization of Nucleolar Organizer Regions in Paraffin Embedded Tissues

A modification of the silver colloid technique for staining nucleolar organizer regions in paraffin embedded tissues is described. This modification involves the application of a gold toning step with subsequent gold reduction, if necessary, following incubation of sections in the standard silver colloid solution. Silver stained nucleolar organizer regions (AgNORs) in toned sections are more sharply delineated when compared to untoned controls. in high grade tumors the addition of the toning step results in significantly higher AgNOR counts due to the ability to discriminate more easily individual AgNORs in argyrophilic aggregates within the nucleus. It is recommended, because of enhanced visualization, that this modification of the silver colloid technique be used in studies involving quantification of AgNORs in tissue sections.     

A method for combined C-banding and silver staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

Behavior of the nucleolus organizer regions of the chromosomes in polykaryocytes consisting of micronuclei

The nucleolar organizer regions (NORs) of Chinese hamster chromosomes (clone 237, cell line BIId-ii-FAF28) were studied in mononuclear cells and polykaryocytes induced with colcemid. The chromosomes with NORs were marked as 1, 2, 3, 4. The activity of NORs in mononuclear cells was higher in chromosomes 1, 2, 3. The associations of NORs were observed between chromosomes I and 2 (3% of all metaphases). In polykaryocytes the chromosomal pairs 1, 2, 3 showed different NOR activity in different metaphases. The associations of NORs in pairs of chromosome I were found in 51.3% of cases. The associations of NORs in pairs of chromosome 2 were observed in 7.5% of cases. This method may be used for the estimation of association potency of NORs in chromosomes.