Importance of interphase nucleolar organizer regions in tumor pathology.

The importance of the distribution of silver-stained nucleolar organizer regions (Ag-NORs) in interphase nuclei for diagnostic and prognostic purposes in tumor pathology has been reviewed. The available data demonstrated that interphase Ag-NOR evaluation may be of help in distinguishing malignant from hyperplastic or normal cells. On the other hand, there is increasing evidence that a relationship exists between the quantity of interphase Ag-NORs and the prognosis of malignant tumors: the greater the number of interphase Ag-NORs, the worse is the prognosis. This can be explained by the observation that the interphase Ag-NOR quantity is strictly related to the cell proliferation rate. The procedures used for the measurement of the interphase Ag-NOR quantity are also critically discussed.     

太平洋鳕染色体核型及银染分析

以野生太平洋鳕为材料,采用植物血球凝集素（PHA）及秋水仙素体内注射法,取头肾细胞经低渗、固定后,常规空气干燥法制备染色体标本,并对其染色体核型进行了分析。结果表明,太平洋鳕的二倍体染色体数目为2n=46,核型公式为:2n=8m+6sm+20st+12t,NF=60,即有4对中部着丝点染色体、3对亚中部着丝点染色体、10对亚端部着丝点染色体和6对端位着丝点染色体,染色体臂数为NF=60;染色体经银染后,Ag-NORs在不同间期细胞中表现出多态性,数目为13,其中2个Ag-NORs的频率最高（82%）;在分裂相中,具有1个Ag-NORs的频率最高（87.1%）,且在第12对亚端部着丝点染色体的一条带有明显的次缢痕,为Ag-NORs所在区域,并未发现Ag-NORs联合现象及性别相关的异型染色体。       

Ag-NOR technique in fine needle aspiration cytology of salivary gland masses.

Because of their complexity, salivary gland lesions are often difficult to identify correctly with fine needle aspiration cytology. To see whether the Ag-NOR staining technique for nucleolar organizer regions would be useful in this respect, we studied a series of smears from benign and malignant salivary gland lesions. The smears, previously treated with Papanicolaou and May-Grünwald-Giemsa stain, were destained and restained with Ag-NOR silver. The correlation between the cytologic-histologic diagnosis and the number of Ag-NORs in benign (sialadenitis, pleomorphic adenoma, oncocytoma and Warthin's tumor) and malignant lesions (adenoid cystic carcinoma, adenocarcinoma, carcinoma ex pleomorphic adenoma and squamous carcinoma) was statistically significant (P = less than .05). The Ag-NOR technique appears useful in the diagnosis of salivary gland lesions. One great advantage is that previously stained slides can be reused for silver staining, thus providing an excellent guide to the diagnosis, especially in doubtful cases and when corresponding histologic specimens or extra unstained slides are unavailable.     

Nucleolar Organizer Regions In Pituitary Gland Adenomas

Nucleolar organizer regions are DNA segments concerned with the synthesis of ribosomal RNA (rRNA). Some specific proteins related to NORs can be demonstrated by a silver technique and are then referred to as Ag-NORs. The mean number of Ag-NORs per cell has been used as a marker of cell proliferation in recent years and there is some evidence to suggest that Ag-NOR counts can be used to differentiate between normal and neoplastic cells. Paraffin block sections of 42 pituitary gland adenomas and 24 normal adult pituitary glands have been examined using the Ag-NOR staining technique. Touch imprints of nine adenomas and 12 normal adult pituitary glands were also examined using the same Ag-NOR staining technique. Using light microscopy at x2000 magnification, the Ag-NORs were located as black dots in the cell nuclei and the mean number of Ag-NORs per cell was determined. No significant difference was observed between blocked sections and imprints of adenomas or between imprints and paraffin sections of normal glands. However, a significant difference was observed between the mean number of Ag-NORs in pituitary gland adenomas and normal glands in paraffin block sections and touch preparations (P less than 0.001). The modified Ag-NOR technique can be a useful aid in the differential diagnosis between normal pituitary gland tissue and pituitary adenomas, especially during surgical operations.     

Differential rRNA Genes Expression in Hexaploid Wheat Related to NOR Methylation

Ribosomal RNA genes expression was analysed in 18 Portuguese bread wheat accessions (Triticum aestivum L. em Thell.), and the number of argyrophilic-nucleolar organiser regions (Ag-NORs) per cultivar was scored. Ten accessions presented six Ag-NORs per metaphase and six nucleoli per interphase, and eight accessions presented four Ag-NORs per metaphase and four nucleoli per interphase. Fluorescent in situ hybridisation with the 45S rDNA sequence pTa71 and genomic DNA from Aegilops tauschii (2n = 2x= 14, DD) confirmed the Ag-NOR location and identified the six satellited chromosomes as being the chromosome pairs 1B, 6B and 5D. The methylation pattern of the NOR region was studied by Southern blot using pTa71 as probe, which represented a complete rDNA unit of bread wheat. DNA digestions performed by MspI and HpaII resulted in different patterns revealing the high level of cytosine methylation at their recognition sequences. The total percentage values of NOR methylation indicated that wheat accessions with a maximum of four Ag-NORs were more heavily methylated at the NOR region than accessions with a maximum of six Ag-NORs.     

NORs and statellite associations in a family with 13/14 translocation

Summary The distribution and size of Ag-NORs and the frequency of satellite associations was investigated in a family where the mother and a son were 13/14 translocation carriers. In cells with good quality silver impregnation and G-banding, Ag-NORs were constant per subject in number and distribution, while Ag-NOR size varied from cell to cell. The father had the maximal number Ag-NORs (10). The mother's translocation chromosome, free chromosome 13 and both chromosomes 22 were Ag-NOR negative and these were transmitted to the children. The mean number of associations per cell for a particular subject was positively correlated with the subject's characteristic number of Ag-NORs. In this family, the positive correlation was also present between mean Ag-NOR sizes of acrocentric homologue chromosome pairs and their coefficient of association. No biological mechanism compensating for the absence of active NORs was demonstrated for this family.     

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.     

Activity of nucleolus organizer regions (NORs) in embryonic livers from twin minks]

Ag-NOR patterns were studied in hepatocytes from nine mink embryo siblings, including a pair of monochorionic (presumably monozygotic, MZ) twins. Both the number and the size of Ag-NORs per cell were found to be identical in MZ twins. All the other sibs had the patterns different from each other and from the MZ ones. The conclusion is that the NORs activity is a strongly inherited character and the Ag-NOR pattern can be used as a reliable genetic marker to distinguish the twin zygosity.     

Chromosome banding in salmonid fish: nucleolar organizer regions in Salmo and Salvelinus

Chromosome banding patterns obtained by silver staining (Ag-NORs) were analyzed in three species of Salmo (rainbow, brown trout, and Atlantic salmon) and three species of Salvelinus (brook trout, lake trout, and arctic char). In rainbow trout and Atlantic salmon the Ag-NORs were found at the secondary constrictions of a single chromosome pair, while in brown trout the Ag-NORs were found on the short arms of one or two of the two longest subtelocentric or acrocentric chromosome pairs. The location of the Ag-NORs was multichromosomal in the three Salvelinus species, occurring on one or both members of four to six different chromosome pairs in different individuals. The Ag-NOR sites were on the short arms of some acrocentric pairs and at the telomeres of other acrocentric pairs and one or two metacentric pairs. Chromomycin A3 positive bands were found at the same sites as the Ag-NORs in all species. In the species with multichromosomal location of Ag-NORs, polymorphisms in the size and location of the NORs were extremely common, so that almost every individual fish had a different pattern of Ag-NOR sites.     

Numerical variation of nucleolar organizer regions after silver staining in domestic and wild Suidae (Mammalia)

Selective silver staining was used to investigate the cellular distribution of numbers of nucleolar organizer regions (NORs) in domestic pigs (Sus scrofa) of eight different breeds, the European wild boar (S. scrofa scrofa), Indonesian wild boar (S. scrofa vittatus), Javan warty pig (S. verrucosus), Sulawesi warty pig (S. celebensis), and pigmy hog (S. salvanius). In the domestic pig as well as in the wild (sub)species of Sus, actively transcribing ribosomal RNA genes were found to be present in the secondary constrictions of chromosome pairs 10 and 8. Chromosomes 10 were consistently Ag-positive. Chromosomes 8 less frequently showed Ag-NORs, resulting in different mean numbers of Ag-NORs per individual animal. Mean Ag-NOR numbers per breed or (sub)species were generally higher in the wild representatives of Sus than in the domestic breeds. The highest mean numbers of Ag-NORs were observed in the Meishan breed and in S. celebensis and S. salvanius. The Meishan breed appears to be conservative in Ag-NOR staining pattern, being more comparable to the Asian wild Suidae than to the European breeds.     

Number and distribution of silver-stained nucleolar organizer regions and evolutionary relationships in domestic pigs

Variation in the numbers of silver-stained nucleolar organizer regions (Ag-NORS) were examined in 36 breeds of domestic pig of different geographic origins and five subspecies of wild boar. The relationship between Ag-NORs and evolution of domestic pigs was investigated. In all pigs observed, Ag-NORs were localized on the secondary constriction of chromosomes 10 and 8. The mean Ag-NOR numbers varied from 2.0–4.0, and decreased gradually with the different geographical distribution from south to north in China and from east to west in Europe. This regular change was caused mainly by the differences of frequency in chromosome 8 Ag-NOR type and was closely related to the evolution of domestic pig breeds.     

一例12号染色体臂间倒位的细胞遗传学研究

小麦主要经济性状大多属数量性状,是受 微效多基因控制的。为了探索小麦常用亲本配 制组合时各主要经济性状的遗传变异规律,于 1980年选用了江苏、浙江两省普遍采用的UP 301、泰山1号、908、早白、白壳早、短秆早6 个品种作为杂交亲本,研究并分析了主要经济 性状的遗传参数,为有效地选配亲本,提高育种 成效,提供一些理论依据。     

Location of rRNA genes in three inbred strains of rat and suppression of rat rRNA activity in rat-human somatic cell hybrids

Nucleolus organizer regions (NORs) of rat chromosomes were stained by the Ag-AS method. The Ag-NORs were found on chromosomes 3, 11 and 12 in the ACI, Wistar Brown and Wistar Lewis inbred strains of rat. The size of the Ag-NOR on each pair of chromosomes varied from strain to strain. Rat-human somatic hybrid cells that retained human and lost some of the rat chromosomes had no Ag-NOR on rat chromosomes 3, 11 or 12. Since NORs can be Ag-stained only if their 18 + 28S rRNA genes are active, the activity of the rat rRNA genes must have been suppressed in the hybrid cells.     

Evaluation of nucleolar organizer region-associated proteins in endometrial pathology.

In the current study the argyrophil staining technique for NOR proteins (Ag-NORs) has been performed on cases of different endometrial lesions, trying to find an aid in differentiating atypical hyperplasia from well differentiated carcinoma in biopsy specimens. We conclude that the Ag-NOR count, even though in endometrial carcinoma is significantly exceeding that of atypical hyperplastic endometrium, could be a misleading discriminator, because of a wide overlap of values in individual cases.     

Semiautomatic quantification of silver-stained nucleolar organizer regions in tissue sections and cellular smears.

Silver staining of nucleoli reveals argyrophilic proteins associated with nucleolar organizer region (Ag-NOR) proteins. Argyrophilic components appear as dots about 1 micron in diameter dispersed throughout the nucleolus (Ag-NOR dots). The count of Ag-NOR dots is a useful index for improving the cancer diagnosis and determination of prognosis. Here we describe software developed on a medium-cost image analyzer in order to evaluate the mean area of NORs and their number relative to an internal reference, the number and areas of clusters of NORs and the area of the nucleus. Statistical analysis of the data was performed during counting. The first application concerned counting NOR dots during mitosis in cell imprints; those counts were 2.3, 15.3 and 55.56 for the metaphase, telophase and interphase, respectively (relative to unitary dots of metaphase cells). In the second application we demonstrated a significant difference in NOR numbers between two groups of prostatic cancers with good and poor prognoses (6.05 +/- 2.79 SD and 7.96 +/- 3.01, respectively; with Student's t test, = 1.999; P = .05).     

Wheat Neocentromeres Found in F1 Triticale × Tritordeum Hybrids (AABBRH<Superscript>ch</Superscript>) After 5-Azacytidine Treatment

The DNA hypomethylation effect of 5-azacytine (5-AC; a cytosine analog) is widely known. This agent has been used for rRNA gene expression studies of Triticeae amphiploids and hybrids regarding rye rRNA genes suppression caused by the wheat nucleolar dominance phenomenon. However, this situation is reverted by 5-AC treatment which activates rye rRNA gene expression as it has been intensively observed in triticale. For nucleolar dominance studies, we produced F1 multigeneric hybrids (AABBRH^ch; 2n = 6x = 42) from crosses between the triticale cultivar ‘Corgo’ (AABBRR; 2n = 6x = 42) and the tritordeum cultivars HT9 and HT31 (AABBH^chH^ch; 2n = 6x = 42). The hybrid seeds were germinated in a low concentration of 5-AC (treatment) and in distilled water (nontreated control plants). Silver nitrate staining performed in one 5-AC-treated F1 hybrid revealed a reduced number of interphase cells with seven nucleoli, metaphases with eight Ag-NORs, and neocentromeres in the long arm of three wheat chromosomes. Nontreated hybrids presented six Ag-NORs per mitotic metaphase cell and a maximum of six nucleoli per interphase because of the 1R Ag-NOR suppression. No neocentromere was found in the control F1 hybrid plants. Both treated and nontreated seedlings were subsequently evaluated by fluorescent in situ hybridization performed with genomic and repetitive DNA probes to identify H^ch and rye genomes, to confirm Ag-NORs location, and to detect inactive rDNA loci. DAPI counterstaining was also helpful for the detection of neocentromeres in the long arm of three wheat chromosomes. This study allowed us to suggest that 5-AC treatment specifically induced wheat neocentromeres in the F1 multigeneric triticale × tritordeum hybrids.     

银杏雌株染色体Ag-NOR的研究

本文以银杏雌株的幼小胚珠为材料,对其体细胞染色体进行了Ag-NOR染色研究。发现其Ag-NOR(随体)的数目均为4个,大小则有同形和异形两种类型。认为NOR(随体)的数目和形态不能作为性染色体的识别特征,并对NOR的杂合性和多态性进行了讨论。 Abstract:In this paper,the Ag-NOR of somatic chromosomes of young ovulum in Ginkgo biloba was studied.It was found that the number of Ag-NORs(satellite)were four constantly,though there were two kinds in size,homoeotype and heterotype.So we considered that the number and morphology of NOR(satellite)can not be used as identification markers of sex chromosome in Ginkgo biloba.In addition,the heterozygosity and polymorphism of NOR are discussed.     

Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.