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.     

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.     

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).     

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.     

Karyotype, DNA Content and Meiotic Behaviour in Five South American Species ofVicia(Fabaceae)

This paper presents the karyotype, DNA content and meiotic behaviourof five species ofViciafrom Argentina (V. macrogramineaBurk.,V.gramineaSM.,V. epetiolarisBurk.,V. pampicolaBurk. andV. nanaVog.).All the species have the same chromosome number and karyotypeformula (2n=14; 6m+4st+4t). Each species, however, displaysa characteristic number and position of the nucleolar organizerregion (NOR) and different sizes of the respective satellites,confirmed by Ag-NOR banding. Moreover, significant differenceswere found in the total chromosome volume (TCV) and DNA contentof the species. Positive correlations between DNA content andTCV, and between DNA content and type of life cycle were alsofound. TCV and DNA content are lower inV. nana(annual) and higherinV. macrograminea(biennial–perennial). The material displayedmarked karyotypic orthoselection, with similar karyotypes inall studied species, even when the overall chromosome size varied.Evolutionary changes in DNA amount are proportional to the relativelength of each chromosome arm, maintaining karyotypic uniformity.Significant differences were found between the meiotic behaviourofV. gramineaand that of the other species.V. gramineahas alower frequency of ring bivalents and chiasmata per cell, andalso has a lower interstitial chiasma frequency. In general,the results are congruent with the morphological data reportedfor these species.Copyright 1998 Annals of Botany Company. Viciaspecies, karyotype, orthoselection, nuclear DNA content, NOR banding, meiotic behaviour.     

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.     

Variability of the nucleolar organizer activity in human lymphocytes via Ag-staining

Summary Chromosomes with Ag staining that varies from one metaphase to the other can be distinguished from those with an Ag-staining that is the same in all metaphases. The intercellular variation of an Ag-NOR can be attributed to many different factors. Whatever the importance of technical factors, they do not seem to account for the large variations in Ag-staining which were observed for each ac. This suggests the existence of a natural intercellular variability of the NOR's activity. The variation of the Ag-stainability of a given NOR, the diversity of Ag-stainings observed on the ten ac of one individual and the differences that exist between individuals raise the question of the existence of a compensation of activity between nucleolar organizers. The study, for each individual, of the mean sum of staining per metaphase reveals that this value is not absolutely constant from one individual to another; in the carriers of Robertsonian fusions it is smaller than in chromosomally normal individuals. The analysis of the transmission shows that inactive NORs remain inactive and that active NORs present a variation in the activity from one generation to the next.     

Ag-NOR staining and in situ hybridization of rDNA in the chromosomes of the South American camelids

The location and frequency of Ag-stained NORs and sites of rDNA hybridization were studied in the chromosomes of the South American camelids. In the four camelids these regions occur distally on chromosomes 18, 21, and 27 and the smallest biarmed elements. Quantitative analysis of NOR distribution showed variations between both cells and species. In llama, guanaco and alpaca the NORs number averaged 6 per cell, this being higher than in vicuña where the average was 3. Relative frequencies of NOR-bearing chromosomes in the four camelids were similar. Yet, in vicuña the virtual absence of NOR sites on one of the smallest biarmed pairs was observed. The rDNA sites assessed in llama and vicuña by in situ hybridization with cloned 18S DNA were coincident with the NOR locations and with the frequencies characteristics for each species. Moreover, varying the exposure time of the autoradiographs, labeling patterns specific for each camelid were observed. Grain counts on individual chromosomes indicated that under our conditions one month exposure is enough to demonstrate all the rDNA sites available in the complement of llama. Conversely, at least two months are necessary to show the total sites existing in vicuña. Most probably this finding reflects the presence of variations in the amount of copies of the ribosomal genes per chromosome.     

Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus)

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.     

癌症患者淋巴细胞核仁形成区活性变异的研究

应用银染-G带复合显示方法研究了肺癌、胃癌、肠癌、乳腺癌患者的外周淋巴细胞核仁形成区(NOR)活性。与正常对照相比,肺癌第15号染色体的Ag-NOR频率及Ag-NOR总频率增加,乳腺癌第14号染色体的Ag-NOR频率减少,胃癌第14号染色体Ag-NOR频率减少而第22号染色体的Ag-NOR频率增加。肠癌未见明显变异。结果提示不同部位的肿瘤具有不同的优势银染型,rRNA基因的表达可能存在肿瘤部位的特异性。     

Frequency of Ag-stained nucleolus organizer regions in the chromosomes of cattle

Sequential Ag NOR/G-band studies were performed on the chromosomes of 33 Austrian Al bulls. The mean number of Ag-NORs per bull karyotype was 6.06. The distribution of Ag-NORs was found to be characteristic for each individual.     

Inheritance of Ag-stainability of nucleolus organizer regions

Summary The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding in cultured lymphocytes from seven children with trisomy 21 and their parents. The observed Ag-NOR patterns were in accordance with chromosomal inheritance except for a slight intraindividual variation which might be explained mainly by technical causes. In two cases the meiotic nondisjunction could be attributed to one of the parents, once to the father, and once to the mother. It is concluded that the Ag-stainability of the NORs is in general a heritable characteristic of the acrocentric chromosomes in maximally activated cells as, e.g., cultured lymphocytes. It may reflect individual differences in the amount of rDNA as well as differences in the capacity for NOR activation.     

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.     

Comparative analysis of the population polymorphism of human silver-staining chromosomal nucleolus organizer regions

The population study of nucleus organizing regions (NOR) activity in individuals of Georgian nationality was carried out using the Ag-method. The results obtained were compared with those for other populations: Russian, Viena-Ulm, Estonian etc. The mean number of active NORs for Georgians is 9.0, which significantly exceeds analogous indexes for the rest of populations, except Viena-Ulm one. The population differences in the distribution of NOR activity and the ability to be stained was found for individual NORs. The lowest activity was registered in chromosome 14, the highest being observed for chromosomes 21 and 13. It is assumed that some mechanism exists that controls the level of general NOR activity in the cell.     

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.     

Ribosomal RNA expression in a mammalian hybrid,the hinny

The expression of nucleolus organizer region (NOR) activity in diploid cells was investigated in a model mammalian hybrid system, the hinny (female ass x male horse), by sequential Ag-NOR and chromomycin A3/distamycin A/DAPI (CDD) staining in lectin-stimulated peripheral blood lymphocytes. In the majority of cases we found non-expression of the horse-derived NOR chromosomes in the hinny. However, in one case there was strong NOR expression on horse-derived chromosome no. 1.     

Chromosomal differentiation of Hyla nana and Hyla sanborni (Anura,Hylidae) with a description of NOR polymorphism in H. nana

Specimens of Hyla nana and Hyla sanborni from a syntopic population were studied cytogenetically. These species are morphologically very similar and are frequently misidentified, confused with each other. Both species had a diploid chromosome number, 2n = 30. However, the karyotypes of H. nana and H. sanborni differed considerably from each other in the number of submetacentric and telocentric chromosomes. The two species also differed in their primary NOR-bearing chromosomes (metacentric pair 13 in H. nana and telocentric pair 12 in H. sanborni). Additional nucleolus organizer regions (NORs) were detected by Ag-NOR staining and FISH in chromosome pairs 1, 5, 6, 12, and 14 in seven specimens of H. nana. Thus, a total of six patterns of NOR were identified. These differences in karyotype and in NOR location allowed the unambiguous identification of syntopic individuals of the two species. However, the chromosomal morphology of both species differed from that reported for populations from other geographic regions, suggesting that a systematic reevaluation of this group of Hyla may be necessary.     

Karyotypic evolution of ribosomal sites in buffalo subspecies and their crossbreed

Domestic buffaloes are divided into two group based on cytogenetic characteristics and habitats: the “river buffaloes” with 2n = 50 and the “swamp buffaloes”, 2n = 48. Nevertheless, their hybrids are viable, fertile and identified by a 2n = 49. In order to have a better characterization of these different cytotypes of buffaloes, and considering that NOR-bearing chromosomes are involved in the rearrangements responsible for the karyotypic differences, we applied silver staining (Ag-NOR) and performed fluorescent in situ hybridization (FISH) experiments using 18S rDNA as probe. Metaphases were obtained through blood lymphocyte culture of 21 individuals, including river, swamp and hybrid cytotypes. Ag-NOR staining revealed active NORs on six chromosome pairs (3p, 4p, 6, 21, 23, 24) in the river buffaloes, whereas the swamp buffaloes presented only five NOR-bearing pairs (4p, 6, 20, 22, 23). The F1 cross-breed had 11 chromosomes with active NORs, indicating expression of both parental chromosomes. FISH analysis confirmed the numerical divergence identified with Ag-NOR. This result is explained by the loss of the NOR located on chromosome 4p in the river buffalo, which is involved in the tandem fusion with chromosome 9 in this subspecies. A comparison with the ancestral cattle karyotype suggests that the NOR found on the 3p of the river buffalo may have originated from a duplication of ribosomal genes, resulting in the formation of new NOR sites in this subspecies.     

The protein-synthesizing apparatus of a BHK-21 (C-13) cell culture and of its large-cell clone C-9

Cell line BHK-21 (C-13) and its large cell clone C-9 differ in morphology, karyotype and cultural properties. Clone C-9 is polyploid. It has been shown that C-9 clone cells display 2.5-3-fold excess in the nucleolus organizer region (NOR) in chromosomes, and 2-3 times higher intensity of protein synthesis and of ribosomal material content compared to the original line. Data of sedimentation analysis and of protein synthesis activity of the total ribosomal material in the cell-free translational system from rabbit reticulocytes allow to conclude that the quantity and size of polyribosomes in C-9 cells are higher than in cells of the original line. Apparently, the quantity of NOR-chromosomes reflect the activity of protein translation system of investigated cells.