Polymorphisms of Ag-stained nucleolar organizer regions in man

Summary Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.     

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.     

Clonal analysis of the intercellular variability in the functioning of human chromosomal nucleolus organizer regions

Intercellular variability of NOR activity detected with the aid of Ag-staining of human chromosomes was studied in mass and cloned fibroblast cultures obtained from 3 individuals. The intercellular variability was determined by different staining of one of 10 NORs. According to this trait the heterogeneity of the cell population was discovered in all cloned lines, with this heterogeneity being the same as in the parent cultures. That concerned the number of a variable chromosome and the percentage of the cells with Ag-stained and unstained chromosomes. It is suggested that genetic determination in the progenies of the somatic cells concerns the whole spectrum of potential variability observed in cell populations.     

Silver staining analysis of nucleolar-organizer activity during spermatogenesis of Asellus aquaticus (Crustacea,Isopoda)

The Ag-staining technique was employed to investigate the activity of the nucleolar organizer during spermatogenesis of the isopod crustacean Asellus aquaticus. The most interesting results of this investigation were: (1) The NORs remain continuously Ag-stained for the whole of spermatogenesis until maturation of the sperm, contrary to the situation in the other species so far described; (2) In the gonial mitotic cells the NORs of a single pair, in the meiotic cells of both pairs of chromosomes that have the NORs, are Ag-stained and therefore active; (3) Some of the individuals examined exhibited additional NORs.     

Frequency analyses of active NORs in nuclei of artificially induced triploid fishes

Summary The correspondence between increased numbers of both chromosomal and nuclear NORs and artificially induced triploidy in three fish species (rainbow trout, Oncorhynchus mykiss; common carp, Cyprinus carpio; and tench, Tinea tinea) has been confirmed by CMA₃ fluorescence and Ag-staining. The frequencies of cell nuclei with one, two and three active NORs, as revealed by Ag-staining, has been analyzed statistically to find the minimum cell number which verifies the increased ploidy level. A minimum sample size of about 80 cells exhibiting three active NORs is sufficient to confirm triploidy in all three species and may be of use for categorising other ploidy-manipulated fish species.     

rDNA site number polymorphism and NOR inactivation in natural populations ofAllium schoenoprasum

Nucleolar-organiser activity has been studied by silver staining and byin situ hybridization with an rDNA probe in two populations ofAllium schoenoprasum. One population is monomorphic with NORs and rDNA sites terminal on the short arm of pair 8 in all individuals. The other populations is monomorphic for pair 8 NORs but is also polymorphic for NORs on the long arm of pair 7. All plants in this population carry ribosomal cistrons on both chromosomes of pair 7 but 0, 1 or 2 of these sites can be active in rRNA synthesis. Cis-acting nucleolar-suppression affects the pair 7 locus. We suggest that there has been progressive reduction in the number of NORs during the evolution ofA. schoenoprasum.     

Intercellular NOR-AG variability in man

Summary A new modification of the Ag I technique has been developed using human cultured blood lymphocytes, which involves ultra-violet irradiation of chromosome preparations during incubation in AgNO₃. This technique enables detection in a short incubation time all NORs capable of being stained with silver. A peculiar morphological change in Ag-stainable NORs during the incubation is described, which can be used as a criterion of the completion of Ag staining. With the refined Ag-staining procedure, acrocentric marker chromosomes were studied which showed one or two satellite stalks within the same individual. Ag staining was highly coincident with this variability.     

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.     

Ag-staining of nucleolus organizer regions on human prematurely condensed chromosomes from cells with different ribosomal RNA gene activity

The Ag-staining of the nucleolus organizer regions (NORs) of prematurely condensed chromosomes (PCCs) of human cells showing different degrees of rRNA-gene activity clearly indicates a close correlation between the positive Ag-staining of NORs and the activity of rRNA genes. The Ag-stain, however, seems insensitive to low rates of rRNA synthesis and obviously follows a threshold reaction. Furthermore it was found that the frequency of Ag-positive chromosomes involved in satellite associations in interphase does not differ from that in metaphase.     

Cytogenetics of bisexual/unisexual species of Poecilia. I. C-bands, Ag-NOR polymorphisms, and sex chromosomes in three populations of Poecilia latipinna

Three populations of the North American cyprinodont fish Poecilia latipinna, considered to be one of the progenitor species of the gynogenetic unisexual P. formosa, were analyzed by C-banding and Ag-staining. C-bands were found to be polymorphic, and Ag-staining showed a high degree of variability in both the number and location of nucleolus organizer regions. The C-banding and Ag-staining patterns allow, to a certain extent, to distinguish individual specimens from each of these populations. Females of the three populations were found to have a heteromorphic chromosome pair, which was frequently identifiable with Giemsa staining and always after C-banding. This pair could be interpreted as sex chromosomes of the ZW/ZZ type.     

"Ag-NORs" are not always true NORs: new evidence in mammals

In spite of uncertainty about the biochemical processes involved, silver staining is a widely used technique for assessing the locations of active NORs in eukaryotic genomes in general, and in mammalian genomes in particular. However, following a previous study of hedgehog chromosomes, we present here a second example from two gerbil species (Rodentia, Muridae), which have several clear Ag-positive signals that do not correspond to 28S rDNA clusters. Although this pattern may be characteristic of particular genomes displaying unusual heterochromatic features, our study casts doubt upon the reliability and universality of Ag-staining for detecting active NORs.     

A possible cause of non-disjunction of additional chromosome 21 in Down syndrome

Summary A possible cause of non-disjunction of chromosome 21 in Down Syndromes has been cytogenetically evaluated by examining the parents by Ag-staining technique. In all the cases studied so far, the contributing parents have active ribosomal cistrons on both chromosomes 21 i.e. both chromosomes are stained positively by silver staining. These results show that the active NORs might play an essential role in meiotic non-disjunction. Furthermore, the preliminary results demonstrate that the acrocentric associations of homologous and non-homologous nature involving chromosome 21 are the most frequent in the contributing parent which may further indicate the role of multiple cellular factors affecting the associations in promoting the non-disjunction in addition to active NORs. The possible mechanisms regarding the non-disjunction of chromosome 21 have been described.Presented at the 34th Annual Meeting of the American Society of Human Genetics, Norfolk, VA, USA     

Chromosome banding in Amphibia

The structure of the nucleolus organizer regions (NORs) in mitotic chromosomes, diploid nuclei and spermatogenesis was studied in 260 individual animals from 23 genera of the Anura. The analyses were performed with conventional cytogenetic methods as well as with Ag-staining, GC- and AT-specific fluorochromes (mithramycin, chromomycin A₃, quinacrine) and C-banding. Most of the species have only one pair of NORs in their karyotypes. The majority of individuals of all species exhibited considerable differences in the sizes of their homologous NORs. Most of these heteromorphisms are due to tandem duplications or triplications in one of the two NORs. However, duplicated or triplicated NORs never occur in a homozygous form, but are instead always in combination with a normal-structured NOR in the homologous chromosome. In three animals, a complete deletion of one NOR and its closely associated constitutive heterochromatin was determined. The cytochemistry of the specific NOR-stainings are discussed. The size differences of the Ag-, mithramycin- and chromomycin A₃-stained NORs can be traced to differences in the rDNA content in these NORs.     

黄颡鱼染色体的C─带、Ag—NORs、荧光带和复制带显带的研究

采用Giemas染色、C─带、Ag—NORs、荧光染色和复制带显带的技术对黄颡鱼染色体进行了研究。结果表明,黄颡鱼只有部分的染色体呈现阳性C─带,可分为三类,其中NORs区是染色最深、染色面积最大的区域,为深染居间C─带。其Ag－NORs位于m5q末端。CMA3染色显示NORs区呈现出明亮的荧光。中复制染色体上着丝粒区、端粒区和居间区浅染。发现核仁缢痕、深染居间C─带、Ag—NORs、CMA3明亮区和中复制带浅染NORs区位置基本一致,C─带阳性区和中复制带浅染区具有对应性。     

Characterization of chromosomes and localization of the rDNA locus in the aye-aye (Daubentonia madagascariensis).

The karyotype of a prosimian primate, the aye-aye (Daubentonia madagascariensis), is described. Results from a variety of staining methods (Q-, R-, G-, and C-banding, distamycin A/DAPI and methyl-green/DAPI) are reported. Sites of methylation were visualized using antibodies against 5-methylcytosine. Digestion of aye-aye fixed metaphase chromosomes with the restriction endonuclease HaeIII produced G-banding. No other restriction enzymes tested produced clear G- or C-banding patterns. Ag-staining of the nucleolar organizer regions (NORs) revealed the location of rDNA sites on the short arms of the smallest pairs of acrocentric chromosomes, 13p and 14p.     

Patterns of silver staining in cells of six-day blastocyst and kidney fibroblast of the domestic rabbit

The distribution pattern of silver-NORs was studied in cells of six-day blastocysts and kidney fibroblasts of the rabbit using the Ag-AS technique. At metaphase and interphase there was a binomial distribution of the number of stained sites in both populations but blastocysts had a greater percentage of cells with larger numbers of stained sites. Up to 7 of the 8 chromosomes known to bear NORs were stained in cells from blastocysts while a maximum of 6 were stained in fibroblasts. A significant difference was found between the mean numbers of chromosomal NORs per cell in metaphases from blastocysts and fibroblasts, where they were 4.2 and 3.3 respectively. Similarly, the mean number of NORs in interphase was significantly greater in cells from blastocysts. The distribution of staining on chromosome pair 13 was related to cell type. Significantly more cells in blastocysts than fibroblasts showed staining in this chromosome pair.     

The correlation between silver-staining and DAPI-induced chromosome undercondensation

The silver-staining pattern of Chinese hamster chromosomes that show DAPI-induced undercondensed areas was studied. The Ag-staining was preceded or not by dehistonizing treatments. In both cases silver preferentially precipitates on undercondensed areas. It is suggested that this phenomenon could be compared to the silver-stainability of the active NORs during mitosis when they remain undercondensed. The possible chemical nature of the protein groups responsible for the silver-staining is discussed.     

Population heteromorphisms of Ag-stained nucleolus organizer regions (NORs) in the acrocentric chromosomes of East Indians

Summary The nucleolar organizer regions (NORs) of acrocentric chromosomes in 70 normal East Indians were examined by Ag-staining (NSG) and acridine orange reverse banding (RFA) techniques. The Ag-stainability of NORs was variable from one individual to another but characteristics were constant within each individual. The average modal number of Agpositive NORs per individual was eight. A racial difference in the expression of NORs is suggested. to study the heteromorphism of NORs, the NSG technique was found to be more useful than RFA. Furthermore, it is concluded that there is no direct relationship between a heteromorphism of NORs identified by NSG and that identified by the RFA technique. Quantitative data on these differences is provided. In addition NOR-regions are classified into five sizes namely; very large, large, medium, small, and very small using subjectively defined criteria.     

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.