Comparative cytogenetics among populations of two Bothriurus species (Scorpiones, Bothriuridae)

Aim:The objective of this experiment was to study the nucleolar organizer region (NOR)-banding pattern in Large White Yorkshire (LWY) crossbred and non-descript pigs and finding differences in the number of NORs between animals and between genetic groups.Materials and Methods:The experiment was carried out on 15 females, and 15 males of LWY crossbred and non-descript pigs to study NOR-banding pattern by employing ammoniacal silver staining technique.Results:A total of 63 and 65 number of good metaphases were prepared in LWY crossbred, and non-descript pigs and a total of 168 and 143 number of NORs were detected on the 8th and 10th chromosomes in both genetic groups, respectively. The mean number of NORs per metaphase was 2.67 and 2.20 in LWY crossbred and non-descript pigs, respectively. LWY crossbred pig had high mean number of silver-stained NORs (Ag-NORs) per metaphase compared to non-descript pig. In general, it was observed that the highest frequency of metaphases (%) examined had two number of NORs, while the lowest frequency (%) had four number of NORs. The number of NORs observed per metaphase on secondary constrictions of the 8th and 10th chromosome pair in both genetic groups ranged from 2 to 4. The Chi-square test of significance revealed that the observed frequencies do not differ significantly from the expected frequencies.Conclusion:The results confirmed differences across breeds in occurrence and number of NORs on chromosomes in pigs. The mean numbers of NORs present per metaphase vary between the animals indicating the existence of polymorphism for the number of NORs. A higher number of Ag-NORs were observed on chromosome pair 10 in both the genetic groups. It was concluded that NORs were more morphologically distinct and greater on chromosome pair 10 than on pair 8, which suggests a dominant role of chromosome 10 in the global production of ribosomal RNA.

BackgroundDespite progress in genomic analysis of spiders, their chromosome evolution is not satisfactorily understood. Most information on spider chromosomes concerns the most diversified clade, entelegyne araneomorphs. Other clades are far less studied. Our study focused on haplogyne araneomorphs, which are remarkable for their unusual sex chromosome systems and for the co-evolution of sex chromosomes and nucleolus organizer regions (NORs); some haplogynes exhibit holokinetic chromosomes. To trace the karyotype evolution of haplogynes on the family level, we analysed the number and morphology of chromosomes, sex chromosomes, NORs, and meiosis in pholcids, which are among the most diverse haplogyne families. The evolution of spider NORs is largely unknown.ResultsOur study is based on an extensive set of species representing all major pholcid clades. Pholcids exhibit a low 2n and predominance of biarmed chromosomes, which are typical haplogyne features. Sex chromosomes and NOR patterns of pholcids are diversified. We revealed six sex chromosome systems in pholcids (X0, XY, X1X20, X1X2X30, X1X2Y, and X1X2X3X4Y). The number of NOR loci ranges from one to nine. In some clades, NORs are also found on sex chromosomes.ConclusionsThe evolution of cytogenetic characters was largely derived from character mapping on a recently published molecular phylogeny of the family. Based on an extensive set of species and mapping of their characters, numerous conclusions regarding the karyotype evolution of pholcids and spiders can be drawn. Our results suggest frequent autosome–autosome and autosome–sex chromosome rearrangements during pholcid evolution. Such events have previously been attributed to the reproductive isolation of species. The peculiar X1X2Y system is probably ancestral for haplogynes. Chromosomes of the X1X2Y system differ considerably in their pattern of evolution. In some pholcid clades, the X1X2Y system has transformed into the X1X20 or XY systems, and subsequently into the X0 system. The X1X2X30 system of Smeringopus pallidus probably arose from the X1X20 system by an X chromosome fission. The X1X2X3X4Y system of Kambiwa probably evolved from the X1X2Y system by integration of a chromosome pair. Nucleolus organizer regions have frequently expanded on sex chromosomes, most probably by ectopic recombination. Our data suggest the involvement of sex chromosome-linked NORs in achiasmatic pairing.

Variation in the number and chromosomal location of nucleolar organizer regions (NORs) was studied in the house mouse, Mus musculus (2n=40). From an origin in Western Asia, this species colonized the Middle East, Europe and Asia. This expansion was accompanied by diversification into five subspecies. NOR diversity was revealed by fluorescence in situ hybridization using 18S and 28S probes on specimens spanning Asia to Western Europe. The results showed that the house mouse genome possessed a large number of NOR-bearing autosomes and a surprisingly high rate of polymorphism for the presence/absence of rRNA genes on all these chromosomes. All NOR sites were adjacent to the centromere except for two that were telomeric. Subspecific differentiation established from the NOR frequency data was concordant with the overall pattern of radiation proposed from molecular studies, but highlighted several discrepancies that need to be further addressed. NOR diversity in M. musculus consisted of a large number of polymorphic NORs that were common to at least two subspecies, and a smaller number of NORs that were unique to one subspecies. The most parsimonious scenario argues in favor of a subspecific differentiation by lineage sorting of ancestral NOR polymorphisms; only the unique NORs would have appeared by inter-chromosomal transposition, except for the two telomeric ones that may have originated by hybridization with another species. Such a scenario provides an alternative view from the one prevailing in most systematic and phylogenetic analyses that NORs have a high transposition rate due to concerted evolution of rRNA genes.

In the present study, we examined the chromosome number and morphology, heterochromatin pattern, and location of the nucleolar organizer region (NOR) and telomeric sequences in Aplastodiscus albofrenatus, A. arildae, A. ehrhardti and A. eugenioi in order to cytogenetically characterize the Aplastodiscus albofrenatus group. The 4 species analyzed in this study had diploid numbers of 2n = 22 and very similar chromosome morphology. These species could be differentiated based on the distribution and amount of heterochromatin and the location of the nucleolar organizer region (NOR). Six of the 8 A. albofrenatus individuals had an NOR polymorphism previously unknown in anurans since only one of the homologs of pairs 1 and 7 was stained. In the other 2 specimens, the NOR occurred on both homologs of pair 7. In A. ehrhardti, pairs 6 and 10 were stained by the AgNOR technique, but only pair 6 was confirmed by in situ hybridization. The NOR was located on pair 10 in A. arildae and on pair 7 in A. eugenioi. In A. albofrenatus and A. arildae, multivalent rings involving NOR-containing chromosomes were observed during prophase I of meiosis. The telomeric probe identified the telomeres in all species and also centromeric regions in the chromosomes of A. albofrenatus and A. arildae, which were coincident with centromeric heterochromatin. The conserved chromosomal morphology seen mainly in the first 7 pairs among species of the A. albofrenatus group suggests that all of these species probably originated from a common ancestral karyotype and that chromosomal rearrangements resulted in karyotype differentiation, with changes in NOR location, as well as telomeric and heterochromatin dispersal.

A human fibrosarcoma line, HT1080-6TG, with a near diploid number of chromosomes, has an average of 7.3 chromosomes with an Ag-stained nucleolus organizer region (NOR). Cells of this line with an increased number of chromosomes have an increased number of Ag-stained NORs. This cell line has been used as the human parent in constructing mouse-human and rat-human hybrids that segregate rodent chromosomes. The hybrid ccell lines, which have 100 or more chromosomes per cell, show a proportionate increase in the number of Ag-stained NORs (means, 11.4--16.8). The frequency of association of acrocentric chromosomes increases in a similar fashion. There is no evidence of inactivation of human NORs in these cells.

The genus Pareiorhina, formerly considered monotypic, was recently described to comprise two new species P. carrancas and P. brachyrhyncha. Karyotype analysis allowed to infer cytotaxonomical diagnostic characters for P. rudolphi and P. brachyrhyncha in the present study. The species showed the same diploid number (2n = 54) and karyotypes with 18 m, 32sm, 4st and 18 m, 30sm, 6st chromosomes, respectively. In addition, we found that the nucleolus organizer regions (NORs) are located on different chromosomes. Localization of NORs has been ascertained by fluorescent in situ hybridization (FISH) and colloidal silver nitrate stain (Ag-NORs). P. rudolphi shows NORs in interstitial position of the long arm of chromosome pair number 11, while P. brachyrhyncha shows subterminal NORs only in the short arm of chromosome pair 25. Occurrence of diploid chromosome number and presence of only one pair of NOR bearing chromosomes makes these species to comprised of most plesiomorphic karyotype of Loricariidae and supports its clustering with the genus Neoplecostomus. The chromosome types and diversification in the location of NORs corroborate the description of the species P. brachyrhyncha.

Apoptosis and expression of argyrophilic nucleolus organizer regions in epithelial neoplasms of the larynx

The objective of this study was to determine the variation in the number and size of nucleolar organizer regions (NOR) in the chinchilla karyotype. The study was performed with 12 standard chinchillas of two different lines. NORs were visualized on chromosome preparations by Ag-NOR silver staining. Four NOR size classes (I-IV) were determined on the basis of the results obtained, ranging from 0.070 (class I) to 0.229 (class IV). The mean NOR size was 0.144 µm² (SD=0.031 µm²) and fell within class II (from 0.101 to 0.150 µm²). Differences in the relative silver deposit area between the NOR-bearing pair of chromosomes were significant for 3 animals (P < 0.01) and for 1 animal (P < 0.05). The mean number of NORs in the animals ranged from 1.4 to 2.0 (SD=0.00–0.55). It was lower for chinchillas from central Poland (1.53±0.50) compared to those from southern Poland (1.68±0.48), with no significant differences (P > 0.05). The variation observed in the NOR size and number in the chinchilla karyotype indicates the occurrence of NOR polymorphism in the population.

A sensitive staining method for nucleolar organizer regions (NORs) is described, using blue toning of AgNORs. NORs are loops of DNA which are transcribed into ribosomal RNA. NORs can be demonstrated by staining with silver nitrate, since NOR-associated proteins are argyrophilic, producing structures termed AgNORs. Normal blood lymphocytes were stained with both methods. The number and resolution of NORs increased 2-3 times by blue toning (30 mmol/l FeCl3, 11 mmol/l potassium hexacyanoferrate(III), and 33 mmol/l oxalic acid) compared with silver staining. A significant difference in the number of NORs was noticed between silver-stained and blue-toned cells (P < 0.001). The blue toning technique thus appears to be more sensitive in detecting NORs than the AgNOR method and may prove a useful alternative for applications in histopathology.

Nucleolus Organizer Regions (NORs) are the specific chromosome sites where ribosomal genes are located and highly expressed. We have applied the AgNOR staining to identify the distribution of NORs in the chromosomes of Korean Cattle. We have also studied the NORs pattern on the cells originated from different breeds, tissues and sex. Peripheral blood from forty-four Korean Cattle and Holstein was cultured for chromosme preparation. The fibroblast culture from biopsied ear skins was also conducted for chromosome analysis. The distribution of NORs was analyzed by sequential Ag staining and G-banding on metaphases of the cells. In Korean Cattle, the NORs are localized on the telomeres of the five chromosome pairs number 2, 3, 4, 11 and 28. The number of NORs per metaphase ranged from 2 to 10 giving a mean value of 5.6. The number of NORs per cell varied among individuals and cells within same individual. The size of NORs also differed in NO-chromosomes. The number of NORs was significantly different between Korean Cattle and Holstein, fibroblasts and lymphocytes, and male and female. However, the distribution and frequency of NORs were similar among the cells regardless of breeds, tissues, and sex.

Background and Aim:Calfhood disease is an important problem in dairy farming that could cause significant effects on heifer survival and productivity and has economic and welfare effects. Total protein concentration in the blood serum could be one of the predictors of bovine respiratory disease (BRD) in newborn calves. The number of active nucleolus organizers could be used to assess the viability of the protein synthesis system in cells and tissues. We aimed for a comparative assessment of the dynamics of the main indicators of protein metabolism and nucleolus organizer regions (NORs) activity in the lymphocytes of healthy calves (Group I) and calves with BRD (Group II) during the 1st month after birthMaterials and Methods:This study included 30 calves of the red-motley Holstein breed. Venous blood samples were taken from all calves on the 1st, 7th, 14th, and 28th days after birth. Quantitative analysis of total protein (Serum total protein [STP]), immune globulin (Serum immune globulin [SIg]), urea, and creatinine in serum and transcriptionally active chromosome NORs in the interphase nuclei of lymphocytes was conducted using receiver operating characteristic analysis and factor analysis.Results:In Group I, the STP levels decreased during the 1st month of life, and in Group II, the STP levels were variable. The STP levels in both groups remained within the reference intervals. During the first 2 weeks after birth, the calves’ SIg fluctuated within the statistical error limits and did not significantly differ between the groups. On the 28th day, SIg increased in both the groups (by 42.8% for Group I and 33.7% for Group II). The creatinine concentration showed a decrease but did not go beyond the range of reference values. Urea concentration in Group I markedly decreased and remained below the reference values; it did not change in Group II over the entire observation period. The number of NORs in 1-day-old calves did not significantly differ between the groups and amounted to 2.43 in Group I and 2.59 in Group II. A significant increase in the number of active NORs was found in calves in both groups at the ages of 14 and 28 days. Early BRD predictors (at 1-14 days) could not be identified among the studied indicators. The urea and creatinine concentrations and the NOR activity on day 28 after birth could be late BRD predictors. Protein metabolism in the newborn calves’ organisms is regulated by three types of factors: Maintenance of a constant protein concentration in the plasma, protein decomposition, and de novo synthesis.Conclusion:There were no observed significant differences in the protein metabolism values and dynamics of indicators between healthy calves and calves with developed BRD. Alterations in the studied characteristics are the result, but not the cause of BRD. The increase in active NORs under BRD could be a favorable forecasting indicator. Protection against foreign protein and genetic material is a more important task for the organism than ensuring growth processes during the neonatal period.

Twenty confirmed cases of childhood neuroblastoma diagnosed over six years were reviewed and classified according to the subtyping proposed by Shimada et al. The tumours were stained using a silver colloid method for nucleolar organiser regions (NORs), and the mean number of NORs for every 200 cells was calculated. The correlation between the mean number of NORs and histology and survival was studied. There was a significant correlation between the mean numbers of NORs and differentiation, and with the mitosis-karyorrhexis index (MKI) in the stroma poor group (p = 0.01-0.001). A trend to increased survival with decreased numbers of NORs was observed in the study group as a whole (rank order of correlation = -0.57, p = 0.05-0.02). It is suggested that mean number of NORs is of prognostic value in neuroblastomas.

Sixteen cases of malignant brain tumours comprising 6 anaplastic astrocytomas, 3 glioblastoma multiforme, 1 medulloblastoma and 6 metastatic brain tumours were investigated independently by a silver colloid method for nucleolar organizer regions (NORs) and an immunohistochemistry using a monoclonal antibody against a nuclear antigen. Ki-67, in proliferating cells. The correlation between the mean number of NORs and the percentage of Ki-67 labelled cells (Ki-67 labelling index) was examined. In addition, four normal brain tissue samples without neoplastic cells were stained for NOR. The mean number of NORs in these malignant brain tumours was significantly greater than that in normal astrocytes (p less than 0.001). Moreover, both the mean number of NORs and the Ki-67 labelling index in metastatic brain tumours were significantly greater than those in high-grade gliomas (p less than 0.001). The Ki-67 labelling index and the mean number of NORs in malignant brain tumours including metastatic brain tumours were found to be linearly related (r = 0.86). These results suggest that the proliferative potential of malignant brain tumours could be evaluated by NOR score as well as Ki-67 labelling index and that such indices provide clear discrimination between high-grade gliomas and metastatic brain tumours.

Aim. To study the activity of nucleolar organizer regions (NORs) in different Ukrainian cattle breeds in terms of their apparent activity status in silver stain and possible relation with milk productivity. Methods. Chromosome prepara- tions using lymphocytes from the peripheral blood of 90 cows of different breeds were used in the study. NOR activity was determined by visual evaluation of concentrations of silver precipitation on NORs in individual chromosomes. A 50 % silver nitrate solution was used to stain chromosome preparations. NORs were detected as dark spots on telomeres of the corresponding chromosomes. Results. The cytological analysis of chromosome preparations from lymphocytes of first lactation cows detected NOR polymorphism in Ukrainian Red-and-Motley dairy cattle (URM), Ukrainian Black-and-White dairy breed (UBW), and hybrid cows, obtained by crossing Ukrainian Red-and-Motley dairy breed and Montbeliarde bulls (URM × M). First lactation cows of URM and UBM had higher or the same inci- dence of cells with four (29.8 and 30 %) and five (17.1 and 19.5 %) NORs, while in URM × M cows the incidence of cells with the same number of NORs was almost twice lower; cells with 7 and 8 NORs exceeded a similar index for other investigated breeds almost twice (2.5 against 4.5 % and 2.0 against 4.2 %). The highest level of chromosomal aberrations (CA) was observed in the group of animals with medium number (2 to 3 NORs per cell), and the lowest – in the group with a high number of NORs (from 6 to 7) with a reliable intergroup difference (p &lt; 0.01). NOR activity was the highest in the group of animals of local origin (URM × M) with a milk yield over 7,000 kg in 305 days of the first lactation and the lowest in the UBW cows with a milk yield of 4–5,000 kg during the first lactation. Conclusions. We determined the differences in the activity of nucleolar organizers between the investigated groups of cows of dairy breed. URM × M hybrids reliably (р ≤ 0.05) exceeded dairy UBW cows by this index. No statistically significant different was found between other investigated groups of animals by this trait. Higher dairy productivity was found in the animals with higher frequency of NORs in the chromosomes of metaphase cells. In our opinion, the number of active NORs demonstrates relative variability between their number and the rate of protein synthesis, required to implement the productivity traits of the investigated animals.

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.