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.     

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.     

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.     

High Ag-NOR-site variation associated to a secondary contact in brown trout from the Iberian Peninsula

The analysis of nucleolar organizer regions (NORs) using silver (Ag-) staining and in situ hybridization (ISH) in brown trout (Salmo trutta) from various river basins in the Iberian Peninsula revealed high variation in the number and location of NORs. A total of 17 different Ag-NOR sites were revealed in 10 different chromosome pairs. Three different Ag-NOR patterns clustered by river basins and strongly associated to the internal transcribed spacer 1 (ITS1) variation were detected. The main variability in NOR-sites was found in a secondary contact between two divergent lineages of brown trouts at Duero basin. Our results confirmed the abrupt break in the spatial distribution of genetic variation of brown trout populations previously reported at Duero basin. We hypothesize that NOR-site variation might be a consequence of hybridization between divergent lineages of brown trouts and that NORs could play a major role in the maintenance of a hybrid zone in Duero basin via post-zygotic isolation mechanisms.     

Karyotypic characterization and genomic organization of the 5S rDNA in <Emphasis Type="Italic">Polypterus senegalus</Emphasis> (Osteichthyes,Polypteridae)

Polypteridae (Cladistia) is a family of archaic fishes, confined to African freshwaters. On account of their primitiveness in anatomical and morphological characters and mosaic relationships among lower Osteichthyans fishes, they constitute an important subject for the study of evolution in vertebrates. Very little is known about the karyological structure of these species. In this article, a cytogenetic analysis on twenty specimens of Polypterus senegalus (Cuvier, 1829) was performed using both classical and molecular techniques. Karyotype (2n = 36; FN = 72), chromosome location of telomeric sequences (TTAGGG) _n , (GATA)₇ repeats and ribosomal 5S and 18S rRNA genes were examined by using Ag-NOR, classical C-banding, CMA₃ staining and FISH. Staining with Ag-NOR showed the presence of two GC rich NORs on the p arm of the chromosome pair no. 1. CMA₃ marked all centromerical and some (no. 1 and no. 14) telomeric regions. FISH with 5S rDNA marked the subtelomeric region of the q arm of the chromosome pair no. 14. FISH with 18S rDNA marked the telomeric region of the p arm of the chromosome pair no. 1, previously marked by Ag-NOR. (GATA)₇ repeats marked the subtelomeric regions of all chromosome pairs, with the exclusion of the no. 1, 3 and 14. Hybridization with telomeric probes (TTAGGG) _n showed bright signals at the end of all chromosomes. After cloning, the 5SrDNA alignment revealed an organization of sequences made up of two different classes of tandem arrays (5S type I and 5S type II) of different lengths.     

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.     

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.     

Chromosomal Mapping and Molecular Characterization of Ribosomal RNA Genes in Lebias fasciata (Teleostei, Cyprinodontidae)

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.     

A Population Analysis of the Structure and Variability of NOR in Salmo Trutta by Ag, CMA3 and ISH

An analysis of the variation in the number and location of rDNA genes has been carried out in two populations of brown trout (Salmo trutta) from Poland by using Ag and CMA₃-staining, and rDNA in situ hybridisation. We observed an interindividual variation in arm number with NF = 100, 101, and 102. This variation was connected with the size polymorphism of the short (NOR-bearing) arm of the chromosome pair 11. The population studied showed a multichromosomal distribution of active NORs. Atypical Ag-NORs consisted of rDNA genes, as evidenced by rDNA-ISH. In addition to individuals with standard NORs, specimens with extra NORs as well as others with only one active NOR and single interphase nucleolus were observed. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Comparative chromosomal studies on two minnow fish, Phoxinus phoxinus (Linnaeus, 1758) and Eupallasella perenurus (Pallas, 1814); an associated cytogenetic-taxonomic considerations

The present work provides new data on the banding pattern of two cyprinid fish species Phoxinus phoxinus and Eupallasella perenurus from Poland. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the karyotypes. Both of the species karyotypes of 2n=50 were characterised by one pair of acrocentric chromosomes, the largest in the set, and by two pairs of NOR-bearing chromosomes. In the chromosome set of Ph. phoxinus Ag-stained NORs were located on telomeres of two metacentric and two submetacentric chromosomes, but in most metaphases only one of the two homologous was observed. The karyotype of E. perenurus was characterised by Ag-NOR regions at a telomeric position on the shorter arm of two submetacentric chromosome pairs. In most metaphases only three NOR-bearing chromosomes were observed. In both investigated species the location of the A₃ positive signals corresponded with the location of Ag-stained NORs and these sites were associated with heterochromatin shown as C-bands. The results of cytogenetical studies on other related, mainly the North American phoxinins, species are compared and discussed.     

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.     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.     

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.     

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。       

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.     

Frequency and non random distribution of silver-staining NORs on acrocentric chromosomes from neonate and adult human lymphocytes

The NORs frequency in a group of newborns and adults was determined by the gelatine silver staining technique. A higher number of Ag-NORs (χ² test, p<0.01) was found in adults than in newborns. The lack of correlation between cell proliferating rate index (PRI) and frequency of Ag-NORs let us suppose that the decrease of Ag-positive NORs in neonates could probably be due to factors different from cell kinetics. A non random distribution of Ag-NORs on the acrocentric chromosomes was also demonstrated: chromosome 21, in particular, showed the highest frequency, while chromosome 15, the lowest.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

Localization of rRNA genes in Phyllostomidae bats reveals silent NORs in Artibeus cinereus

We analyzed the nucleolus organizer regions (NORs) of thirteen bats from genera Phyllostomus, Phylloderma, Trachops, Tonatia, Sturnira, Platyrrhinus, Artibeus and Glossophaga. We used silver staining and FISH with rDNA probe. Nine species had only one Ag-NOR-bearing chromosome pair. Artibeus lituratus, A. jamaicensis and A. fimbriatus presented multiple Ag-NORs located in the short arms of pairs 5, 6 and 7, and an additional mark in the long arm of one chromosome 5 in A. fimbriatus. Artibeus cinereus showed Ag-NORs in the chromosome pairs 10 and 13. The chromosomal location of rRNA genes using FISH agreed with the number and position of NORs in all but one species. In A. cinereus the hybridization signals were seen in three chromosome pairs 9, 10 and 13. This suggests the occurrence of silent NORs in pair 9. Differences in the size and intensity of the hybridization signals were also observed in the pair 9 of A. cinereus.     

A population analysis of Robertsonian and Ag-NOR polymorphisms in brown trout (Salmo trutta)

An analysis of Robertsonian polymorphism and variation in the number of active NORs has been carried out in several populations of brown trout (Salmo trutta) from Northwestern Spain. The karyotype of this species appears to be soundly established, and essentially no variation has been found in chromosome number. Interindividual and interpopulation variation in arm number was detected, with figures ranging between 100 and 102 among individuals, and between 100.10 and 100.80 among populations. This variation in arm number is solely attributable to the polymorphism of the short arm of the main NOR-bearing pair 11, which can appear from acrocentric to metacentric in different individuals. Most populations analyzed showed the standard distribution of active NORs previously observed in this species. The Miño drainage basin, and specially the Chamoso population, showed a multi-chromosomal distribution of active NORs, with several new locations, always telomeric. In most cases no concordance was observed between previously detected rDNA sites in S. trutta and the new Ag-NOR locations. This fact suggests a transposition mechanism rather than an activation of silent rDNA sites to explain this multichromosomal NOR pattern.