The chromosomes of two Drosophila races: D. nasuta nasuta and D. nasuta albomicans III. Localization of nucleolar organizer regions

In the two Drosophila races D. n. nasuta and D. n. albomicans the nucleolar-organizer regions (NORs) have been localized on metaphase chromosomes by a combined acid treatment and silver-staining technique. In both Drosophila races one NOR is present on the heterochromatic Y chromosomes and another on the microchromosomes. Such a NOR distribution has not yet been reported in Drosophila. It is suggested that this distribution has evolved from an original type with NORs on the X and Y chromosomes.     

Human nucleolus organizers: the satellites or the stalks?

A silver-staining technique specific for demonstrating nucleolus organizer regions (NOR) showed that the achromatic stalks of the 10 acrocentric autosomes of the human complement represent the NORs. Some variability in number of stained stalks is observed from cell-to-cell and from individual-to-individual. The silver-stained masses may extend beyond the stalks and cover the satellites, especially in chromosomes with short stalks or minute satellites.     

Cytogenetic Analysis and Chromosomal Characteristics of the Polymorphic 18S rDNA of Haliotis discus hannai from Fujian,China

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)₃ displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.     

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.     

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH)

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA₃-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n=50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA₃-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants – (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA₃-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.     

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 behavior during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.     

NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae)

Chromosomal polymorphism regarding number of NOR sites in the cyprinid fish Chondrostoma lusitanicum was examined using C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ (CMA₃). The analysis of heterochromatic regions allowed a more precise identification of the centromeric regions and the proposal of a revised haploid chromosome formula (7M: 15S: 3A). We describe variability in the number of NOR regions per genome, number of active NOR sites per cell, and relative size of individual NORs. Individuals expressed two or four NOR-bearing chromosomes. Polymorphism was detected in all the populations studied and sex-related differences were not found. The observed chromosomal NOR phenotypes suggest the occurrence of structural rearrangements during the evolutionary process of this diploid leuciscine cyprinid.     

Conservation of chromosomal location of nucleolus organizer in American marsupials (Didelphidae)

The distribution and expression of nucleolus organizer regions (NORs) were analyzed in seven species of marsupials representative of the three karyotypes (2n = 14, 18 and 22) found in the American family Didelphidae. Analyses comprised silver-staining of NORs and fluorescence in situ hybridization with an rDNA probe. In addition to confirming the variability in number and distribution of NORs in Didelphidae, we demonstrated the conserved location of NORs on one autosome pair in the three karyotypes. In Monodelphis domestica (2n = 18), the NOR on the X chromosome was not inactivated in females.     

Differences in the location of nucleolus organizer regions in European vespertilionid bats

The karyotypes of European vespertilionid bats are distinguished by only a few, easily detectable differences in their G-banding patterns. Most rearrangements can be identified as Robertsonian translocations. Yet, there are surprising differences in the location of active nucleolus organizer regions (NORs), as revealed by silver staining. The ancestral position of the NOR is considered to be a secondary constriction on chromosome 15, as is the case in the genera Eptesicus, Nyctalus, and Vespertilio and in three of four Pipistrellus species. The remaining genera show multiple NOR sites located on minute short arms close to the centromere. In P. pipistrellus, differences in the location of the NORs correlate with the geographical origin of the animals. Some Myotis species possess NORs on numerous chromosomes and show great interindividual variability. In addition, two sibling species, M. brandtii and M. mystacinus, show completely different NOR locations.     

Sequential silver staining and hybridization in situ on nucleolus organizing regions in human cells.

Chromosome preparations from eight individuals were first stained with silver nitrate to reveal the nucleolus organizing regions (NORs) and then hybridized in situ with ribosomal RNA. In six individuals the size of the silver-staining regions was positively correlated with the amount of label present after hybridization in situ. Thus the variation in silver-staining intensity among chromosomes was largely explained by variation in the number of rDNA gene copies per NOR. However, in two individuals this correlation was absent, suggesting that other factors can also influence the size of the silver-staining region.     

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.     

六种麻蜥核型的研究

报道麻蜥属(Eremias ,Lacertidae) 6种15个不同居群的染色体核型及银分带核型。丽斑麻蜥（E. argus）、快步麻蜥(E. velox)、敏麻蜥(E. arguta)、密点麻蜥(E. multiocellata)、网纹麻蜥（E. grammica ）的核型一致：2n=38=36I+ 2m,NF=38;虫纹麻蜥（E. vermiculata） 2n=38=12V+2sI+22I+2m, NF=50。中国麻蜥属的核型可分为3个类型：（1）丽斑麻蜥型（2）山地麻蜥(E. brenchleyi)型（3）虫纹麻蜥型。虫纹麻蜥核型演化有两种可能性（1）经历三倍体阶段,并通过罗伯逊易位形成;（2）通过染色体臂间倒位形成,倒位成因可能和天山山脉以及青藏高原的隆起有关。密点麻蜥、快步麻蜥、敏麻蜥、网纹麻蜥、虫纹麻蜥均观察到一对NOR于一对较小染色体对上。雌雄个体中均未发现性异型染色体。Abstract: Based on the Giemsa-dyeing karyotypes and silver-staining bands of 15 populations from different localities in China belonging to 6 species of the genus Eremias , We found all species studied have 19 pairs of chromosomes, the size of chromosomes reduces gradually and there are no marked differences between the arranged pairs of macrochromosomes except the last pair of microchromosome. There are the same karyotype formula as 2n=38=36I+2m with NF=38 in E. argus、 E. multiocellata、 E. velox、 E. arguta and E. grammica; but the karyotype formula of E.vermiculata is different as 2n=38=12V+2sI+22I+2m with NF=50. The NOR are all located on one small pair in female of E. velox, and E. arguta , in male of E. grammica and E. vermiculata ,and in both male and female of E. multiocellata. We have not found two or more than two pairs of NOR. Having one pair of NOR may be common in Genus Eremias and also the trait of Eremias. We speculate that the derivation of the karyotype of E.vermiculata had two possible way: one experienced the stage of triploid, and later the Robertsonian transposal of chromosomes; the other way was through the inversions between the arms on the chromosome and the phenomenon of inversions might occur during or subsequently after the upheaval of the Tibet and Qinghai plateau and the founding of the Tianshan . With regard to the trend of the evolution of chromosomes in the lizards [1], the karyotype of E.vermiculata is more advanced. Making specialties of E. vermiculata will help in building the phylogenic tree of Eremias. In both male and female of the species studied, the heteromorphic sex-chromosomes were not found.     

Comparative cytogenetics in four species of Palinuridae: B chromosomes, ribosomal genes and telomeric sequences

The evolutionary pathway of Palinuridae (Crustacea, Decapoda) is still controversial, uncertain and unexplored, expecially from a karyological point of view. Here we describe the South African spiny lobster Jasus lalandii karyotype: n and 2n values, heterochromatin distribution, nucleolar organizer region (NOR) location and telomeric repeat structure and location. To compare the genomic and chromosomal organization in Palinuridae we located NORs in Panulirus regius, Palinurus gilchristi and Palinurus mauritanicus: all species showed multiple NORs. In J. lalandii NORs were located on three chromosome pairs, with interindividual polymorphism. In P. regius and in the two Palinurus species NORs were located on two chromosome pairs. In the two last species 45S ribosomal gene loci were also found on B chromosomes. In addition, the nature and location of telomeric repeats were investigated by FISH in J. lalandii, P. gilchristi, P. mauritanicus Palinurus elephas, and P. regius (Palinuridae, Achelata), and in Scyllarus arctus (Scyllaridae, Achelata): all these Achelata species showed the (TTAGG)n pentameric repeats. Furthermore, in J. lalandii these repeats occurred in all the telomeres and in some interstitial chromosomal sites, associated with NORs.     

Cytogenetic analysis of five Hypostomus species (Siluriformes, Loricariidae)

In this work, we analyzed the karyotypes of five Hypostomus species. Hypostomus cf. heraldoi, from the Mogi-Gua?u River, had 2n = 72 chromosomes, with a nucleolar organizer region (NOR) in one chromosomal pair. Hypostomus regani, from the Mogi-Gua?u River had 2n = 72 chromosomes with NORs in two chromosomal pairs. Hypostomus sp., from the Mogi-Gua?u River basin, had 2n = 68 chromosomes, with NORs in two chromosomal pairs. Hypostomus aff. agna, from Cavalo Stream, had 2n = 74 chromosomes with NORs in two chromosomal pairs. Hypostomus cf. topavae, from Carrapato Stream, had 2n = 80 chromosomes, with NORs in two chromosomal pairs. Hypostomus species showed marked diversity in the karyotypic formula, which suggested the occurrence of several Robertsonian rearrangements and pericentric inversions during the evolutionary history of this genus. This hypothesis was supported by the occurrence of a large number of uniarmed chromosomes and multiple NORs in a terminal position in most species and may be a derived condition in the Loricariidae.     

Karyotype analysis of Eucinostomus argenteus, E. gula, E. harengulus, and Eugerres plumieri (Teleostei, Gerreidae) from Florida and Puerto Rico

The karyotypes of four gerreids of the western Atlantic Ocean are documented. A diploid chromosome complement of 48 telocentric chromosomes was found in the four species (2N=48t, fundamental number FN=48). No differences were detected either in the number of chromosomes of the standard karyotype, in their karyotype size, or between the karyotypes derived from male or female specimens of any of the species. Chromosome length decreased progressively and slightly from pair 1 to pair 24. The Ag–NOR karyotypes of E. argenteus and E. harengulus were characterized by the position of the nucleolar organizer regions next to the centromere in chromosome pair 1, whereas in E. gula and E. plumieri Ag–NORs were detected in pair 4. The other 46 chromosomes showed a light staining of the centromere with no terminal or intermediate heterochromatic blocks. All Eucinostomus species showed Ag–NORs of similar size, while Eugerres plumieri showed Ag–NORs 10–20% larger than Eucinostomus species. A combination of size and position of the Ag–NORs identified E. gula, while size alone identified E. plumieri. However, the ancestral state for size and position of Ag–NORs could not be established. There was no differential staining of the chromosomes by G-banding. The karyotype of the gerreids appears similar to the hypothetical ancestral karyotype of fish. The phylogenetic relationships among these species could not be established because of the lack of chromosome G-bands. Most likely this indicates a homogeneous distribution of GC nucleotides in the chromosomes.     

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.     

Relationship between interphase AgNOR distribution and nucleolar size in cancer cells

Summary We have studied the relationship between interphase nucleolar organizer region (NOR) distribution and nucleolar size in cancer cells at light-microscopical level. Thirteen cases of formalin-fixed bladder cancer and fifteen cases of methacarn-fixed tumours of different origin were used. Nucleoli of the former cases were stained by Phloxine B and of the latter by Toluidine Blue. Selective visualization of interphase NORs was obtained by carrying out the one-step silver staining reaction for AgNOR proteins (Plotonet al., 1986). The area occupied by Phloxine B- or Toluidine Blue-stained nucleoli and interphase silver-stained NORs was measured by means of an automated image analyser. Both in bladder cancers and in the other tumour lesions nucleolar and interphase AgNOR areas were linearly related (r=0.95 and r=0.96, respectively,P<0.001). The close relationship between the area of nucleoli and that of silver-stained nucleolar structures was maintained even if the silver-staining procedure was prolonged beyond the optimal time length for selective interphase NOR staining. In the latter case, however, single interphase AgNORs were no longet visible within the nucleolar body which was, in fact, homogeneously stained. These data indicate that evaluation of the interphase AgNOR area has the same relevance, in tumour pathology, as whole nucleolar size measurement.     

BrdU and Hoechst 33258 Induced Silver-Stained Dots on Rye Chromosome Arms

By treating the primary roots with BrdU or Hoechst 33258, silver-stained dots occurrednot only in the NORs, centromeres and telomeres, but also in the arms of rye chromosomes.The mechanism of differential silver-staining of chromosomes was discussed.