Nucleolar Organizer Research Articles

Cytogenetic characterization of Hypostomus soniae Hollanda-Carvalho & Weber, 2004 from the Teles Pires River, southern Amazon basin: evidence of an early stage of an XX/XY sex chromosome system.

In the present study, we analyzed individuals of Hypostomus soniae (Loricariidae) collected from the Teles Pires River, southern Amazon basin, Brazil. Hypostomus soniae has a diploid chromosome number of 2n = 64 and a karyotype composed of 12 metacentric (m), 22 submetacentric (sm), 14 subtelocentric (st), and 16 acrocentric (a) chromosomes, with a structural difference between the chromosomes of the two sexes: the presence of a block of heterochromatin in sm pair No. 26, which appears to represent a putative initial stage of the differentiation of an XX/XY sex chromosome system. This chromosome, which had a heterochromatin block, and was designated proto-Y (pY), varied in the length of the long arm (q) in comparison with its homolog, resulting from the addition of constitutive heterochromatin. It is further distinguished by the presence of major ribosomal cistrons in a subterminal position of the long arm (q). The Nucleolus Organizer Region (NOR) had different phenotypes among the H. soniae individuals in terms of the number of Ag-NORs and 18S rDNA sites. The origin, distribution and maintenance of the chromosomal polymorphism found in H. soniae reinforced the hypothesis of the existence of a proto-Y chromosome, demonstrating the rise of an XX/XY sex chromosome system.

Chromosomal inheritance of parental rDNAs distribution pattern detected by FISH in diploid F1 hybrid progeny of Cobitis (Teleostei, Cobitidae) species has non-Mendelian character.

This study was conducted to describe the major and the minor rDNA chromosome distribution in the spined loach Cobitis taenia (2n = 48) and the Danubian loach Cobitis elongatoides (2n = 50), and their laboratory-produced diploid reciprocal F1 hybrid progeny. It was tested by fluorescence in situ hybridisation (FISH) whether the number of 28s and 5s rDNA sites in the karyotypes of diploid hybrids corresponds to the expectations resulting from Mendelian ratio and if nucleolar organiser regions (NOR)were inherited from both parents or nucleolar dominance can be observed in the induced F1 hybrid progeny. Ten (females) or twelve (males) 28s rDNA loci were located in nine uniarm chromosomes of C. taenia. Two of such loci terminally bounded on one acrocentric chromosome were unique and indicated as specific for this species. Large 5s rDNA clusters were located on two acrocentric chromosomes. In C. elongatoides of both sexes, six NOR sites in terminal regions on six meta-submetacentric chromosomes and two 5s rDNA sites on large submetacentrics were detected. The F1 hybrid progeny (2n = 49) was characterised by the intermediate karyotype with the sites of ribosome synthesis on chromosomes inherited from both parents without showing nucleolar dominance. 5s rDNA sites were detected on large submetacentric and two acrocentric chromosomes. The observed number of both 28s and 5s rDNAs signals in F1 diploid Cobitis hybrids was disproportionally inherited from the two parental species, showing inconsistency with the Mendelian ratios. The presented rDNA patterns indicate some marker chromosomes that allow the species of the parental male and female to be recognised in hybrid progeny. The 5s rDNA was found to be a particularly effective diagnostic marker of C. elongatoides to partially discern genomic composition of diploid Cobitis hybrids and presumably allopolyploids resulting from their backcrossing with one of the parental species. Thus, the current study provides insight into the extent of rDNA heredity in Cobitis chromosomes and their cytotaxonomic character.

Dynamics of morphofunctional activity of blood lymphocytes of Stavropol breed sheep in postnatal ontogenesis and during pregnancy

The aim of the paper was to study the activity of nucleolar organizer zones of blood lymphocytes of Stavropol breed sheep in postnatal ontogenesis and during pregnancy. Blood smears for cytomorphology studies were painted with silver nitrate and subsequent additional coloring with azure and eosin. By measuring the parameters of the nucleus and parameters of the activity of nucleolar organizers (AgNORs), the changes in the morphofunctional state of the protein-synthesizing apparatus of blood lymphocytes in sheep of Stavropol breed in postnatal ontogenesis were evaluated. It was found that newborn lambs’ area of the nuclei of lymphocytes was 76.50 ± 2.30 µm2. By the age of three months, the area of lymphocyte nuclei in sheep significantly increased by 6.08% and amounted to 81.46 ± 2.45 µm2, and did not change significantly until the age of twelve months. The number of units of argentophilic nucleolar organizers in the nuclei of lymphocytes (AgNORs) is from 1 to 4. The average area of AgNORs in the nuclei of lymphocytes is in the range from 73.60 ± 2.45 up to 80.10 ± 2.41 µm2. In sheep of Stavropol breed on the 90th day of pregnancy, the area of the nucleus of lymphocytes was 68.70 ± 2.15 µm2. By 135 days of pregnancy, a significant decrease in the area of the nuclei of the lymphocytes by 14.66% was found. After childbirth, there was a slight decrease in the area of lymphocytes in sheep compared to the previous period of research. During pregnancy, sheep were found to have from 1 to 3 AgNORs with a total area from 0.70 ± 0.11 to 2.93 ± 0.43 µm2. On the basis of the conducted studies, it can be concluded that in postnatal ontogenesis and during pregnancy of sheep, the functional state of lymphocytes changes, this is manifested in changing the parameters of the area of their nuclei, the number and total area of AgNORs.

The human nucleolus organizer regions.

Although the nucleolus was first described in the early 19th century from both animal and plant cells, human nucleoli and particularly the five human nucleolus organizers have not been well characterized. In this issue of Genes & Development, van Sluis and colleagues (pp. 1688-1701) present a detailed molecular analysis of these organizers, which occur on the short arms of five human chromosomes. The near identity of these arms suggests extensive interchromosomal exchange during evolutionary history.

Evolutionary analysis of genus Channa based on karyological and 16S rRNA sequence data

A wide range of diploid number of chromosomes and the body size of Channa congeners are useful combination of characters for studying the factors controlling the body size. In this study, the karyological information was superimposed on the evolutionary tree generated by 16S rRNA mitochondrial gene sequences. Here, the metaphase chromosome complements stained with Giemsa, AgNO3 and CMA3 were prepared from six snakehead murrel fish species collected from northeast India. The diploid chromosome numbers and the fundamental arms of C. aurantimaculata (2n = 52, NF = 98), C. gachua (2n = 56, NF = 84), C. marulius (2n = 44, NF = 58), C. orientalis (2n = 52, NF = 74), C. punctata (2n = 32, NF = 60) and C. striata (2n = 40, NF = 48) were calculated by the analysis of metaphase chromosome complements. Both methods of nucleolar organizer region (NOR) localization, silver nitrate and chromomycin A3, revealed NOR pairs of 1, 2, 3, 1, 4 and 3 in C. aurantimaculata, C. gachua, C. marulius, C. orientalis, C. punctata and C. striata, respectively. The subject species showed primitive type of asymmetrical chromosomes, except the C. punctata. The variation in 2n for C. orientalis (2n = 52, 78) and C. gachua (2n = 52, 78, 104) of a complete haploid set indicates the possibility of either ploidy change in C. orientalis and C. gachua, if we consider 2n = 52 or the Robertsonian rearrangements in different populations of these two species. The chromosome evolution tree was constructed on 16S rRNA ML-phylogenetic tree using ChromEvol 1.3. The analysis of chromosome evolution explained the loss or gain of chromosome, duplications or semiduplications mechanism. For time scaling the chromosomeevolution, the node age of available 16S rRNA gene of Channa species were estimated, which was also used for estimating the time when chromosomal changes occurred in context of geological time-scale.

Human NORs, comprising rDNA arrays and functionally conserved distal elements, are located within dynamic chromosomal regions.

Human nucleolar organizer regions (NORs), containing ribosomal gene (rDNA) arrays, are located on the p-arms of acrocentric chromosomes (HSA13-15, 21, and 22). Absence of these p-arms from genome references has hampered research on nucleolar formation. Previously, we assembled a distal junction (DJ) DNA sequence contig that abuts rDNA arrays on their telomeric side, revealing that it is shared among the acrocentrics and impacts nucleolar organization. To facilitate inclusion into genome references, we describe sequencing the DJ from all acrocentrics, including three versions of HSA21, ∼3 Mb of novel sequence. This was achieved by exploiting monochromosomal somatic cell hybrids containing single human acrocentric chromosomes with NORs that retain functional potential. Analyses revealed remarkable DJ sequence and functional conservation among human acrocentrics. Exploring chimpanzee acrocentrics, we show that "DJ-like" sequences and abutting rDNA arrays are inverted as a unit in comparison to humans. Thus, rDNA arrays and linked DJs represent a conserved functional locus. We provide direct evidence for exchanges between heterologous human acrocentric p-arms, and uncover extensive structural variation between chromosomes and among individuals. These findings lead us to revaluate the molecular definition of NORs, identify novel genomic structural variation, and provide a rationale for the distinctive chromosomal organization of NORs.

Structure of the intergenic spacers in chicken ribosomal DNA

BackgroundRibosomal DNA (rDNA) repeats are situated in the nucleolus organizer regions (NOR) of chromosomes and transcribed into rRNA for ribosome biogenesis. Thus, they are an essential component of eukaryotic genomes. rDNA repeat units consist of rRNA gene clusters that are transcribed into single pre-rRNA molecules, each separated by intergenic spacers (IGS) that contain regulatory elements for rRNA gene cluster transcription. Because of their high repeat content, rDNA sequences are usually absent from genome assemblies. In this work, we used the long-read sequencing technology to describe the chicken IGS and fill the knowledge gap on rDNA sequences of one of the key domesticated animals.MethodsWe used the long-read PacBio RSII technique to sequence the BAC clone WAG137G04 (Wageningen BAC library) known to contain chicken NOR elements and the HGAP workflow software suit to assemble the PacBio RSII reads. Whole-genome sequence contigs homologous to the chicken rDNA repetitive unit were identified based on the Gallus_gallus-5.0 assembly with BLAST. We used the Geneious 9.0.5 and Mega software, maximum likelihood method and Chickspress project for sequence evolution analysis, phylogenetic tree construction and analysis of the raw transcriptome data.ResultsThree complete IGS sequences in the White Leghorn chicken genome and one IGS sequence in the red junglefowl contig AADN04001305.1 (Gallus_gallus-5.0) were detected. They had various lengths and contained three groups of tandem repeats (some of them being very GC rich) that form highly organized arrays. Initiation and termination sites of rDNA transcription were located within small and large unique regions (SUR and LUR), respectively. No functionally significant sites were detected within the tandem repeat sequences.ConclusionsDue to the highly organized GC-rich repeats, the structure of the chicken IGS differs from that of IGS in human, apes, Xenopus or fish rDNA. However, the chicken IGS shares some molecular organization features with that of the turtles, which are other representatives of the Sauropsida clade that includes birds and reptiles. Our current results on the structure of chicken IGS together with the previously reported ribosomal gene cluster sequence provide sufficient data to consider that the complete chicken rDNA sequence is assembled with confidence in terms of molecular DNA organization.

Presence of 15p Marker D15Z1 on the Short Arm of Acrocentric Chromosomes is Associated with Aneuploid Offspring in Mexican Couples.

Variation in the location of the 15p region D15Z1 is recognized as a polymorphism in several human populations. We used high-stringency Fluorescence In Situ Hybridization (FISH) to detect D15Z1 in a Mexican cohort. Here, we report the presence of extra D15Z1 sequences on the p-arm of acrocentric chromosomes other than 15 in two groups of Mexican couples, one with healthy offspring (n = 75) and the other with aneuploid offspring (n = 87), mainly trisomy 21. The additional D15Z1 polymorphism was significantly increased in individuals with aneuploid offspring (26.4%), in comparison to individuals with healthy offspring (14%). The most frequent acceptor chromosome of D15Z1 was chromosome 13p, followed by 14p, and finally, 21p. Our results show an overall frequency of 21.6% of this polymorphism in the Mexican population and suggest that its presence might be associated with the mis-segregation of other acrocentric chromosomes and aneuploid offspring. The high frequency of the polymorphism of the D15Z1 sequence on acrocentric chromosomes other than 15 suggests a sequence homogenization of the acrocentric p arms, related to the important function of the centromere and the nucleolar organization region, which flank satellite III DNA.

Recurrent variation in the active NOR sites in the monkey frogs of the genus Pithecopus Cope, 1866 (Phyllomedusidae, Anura).

Treefrogs of the genus Pithecopus Cope, 1866 exhibit expressive chromosomal homogeneity which contrasts with a high variation frequency of the nucleolus organizer region (NOR) related to the group. Currently, the genus contains eleven species and no chromosomal data are available on P. palliatus Peters, 1873, P. ayeaye Lutz, 1966 and P. megacephalus Miranda-Ribeiro, 1926. Here, we describe the karyotypes of these three species based on Giemsa staining, C-banding, silver impregnation and in situ hybridization (FISH). We were also analyze the evolutionary dynamic of the NOR-bearing chromosome in species of genus under a phylogenetic view. The results indicate that P. palliatus, P. ayeaye, and P. megacephalus have similar karyotypes, which are typical of the genus Pithecopus. In P. palliatus the NOR was detected in the pericentromeric region of pair 9p whereas in P. ayeaye and P. megacephalus we report cases of the multiple NOR sites in karyotypes. In P. ayeaye the NOR was detected in the pericentromeric region of pair 9p in both homologues and additional sites was detected in pairs 3q, 4p, and 8q, all confirmed by FISH experiments. Already in P. megacephalus the NOR sites were detected in pericentromeric region homologues of pair 8q and additionally in one chromosome of pair 13q. A comparative overview of all the Pithecopus karyotypes analyzed up to now indicates the recurrence of the NOR-bearing chromosome pairs and the position of the NORs sites on these chromosomes. We hypothesized that this feature is a result of a polymorphic condition present in the common ancestor of Pithecopus. In such case, the lineages derived from polymorphic ancestor have reached fixation independently after divergence of lineages, resulting in a high level of homoplasy observed in this marker. Our findings help to fill the gaps in the understanding of the karyotype of the genus Pithecopus and reinforce the role of the evolutionary dynamics of the rDNA genes in karyotype diversification in this group.

Vitrification of collared peccary ovarian tissue using open or closed systems and different intracellular cryoprotectants

This study aimed to evaluate different vitrification methods using distinct cryoprotectants (CPAs) for the preservation of collared peccary ovarian preantral follicles (PFs). Ovarian pairs from six females were fragmented and three fragments (fresh control group) were immediately evaluated for morphology, viability, cell proliferation capacity (assessed by quantifying the number of argyrophilic nucleolus organizer regions – NORs), and apoptosis (by the identification of activated caspase-3 expression). The remaining 18 fragments were vitrified using the solid surface vitrification (SSV) method or the ovarian tissue cryosystem (OTC) with 3 M ethylene glycol (EG), 3 M dimethylsulfoxide (DMSO), or a combination of the two (1.5 M EG/1.5 M DMSO). After two weeks, samples were rewarmed and evaluated as described previously. The OTC with any of the CPAs provided a similar conservation of morphologically normal PFs as the fresh control group (75.6 ± 8.6%); however, the SSV was only efficient with DMSO alone (63.9 ± 7.6%). Regarding the viability or cell proliferation, all tested groups provided post rewarming values similar to those observed for the fresh control group, 84.0 ± 2.9% viable cells with 2.0 ± 0.2 NORs. Related to apoptosis analysis, only the OTC with EG (46.7%) and the SSV method with EG (43.4%) or the combination of EG and DMSO (33.4%) provided similar values to those found for the fresh control group (36.7%). Our findings indicate the utilization of a closed system, the OTC, with 3 M EG as the CPA for the vitrification of collared peccary ovarian tissue.

Recurrent variation in the active NOR sites in the monkey frogs of the genus Pithecopus Cope, 1866 (Phyllomedusidae, Anura)

Treefrogs of the genus Pithecopus Cope, 1866 exhibit expressive chromosomal homogeneity which contrasts with a high variation frequency of the nucleolus organizer region (NOR) related to the group. Currently, the genus contains eleven species and no chromosomal data are available on P. palliatus Peters, 1873, P. ayeaye Lutz, 1966 and P. megacephalus Miranda-Ribeiro, 1926. Here, we describe the karyotypes of these three species based on Giemsa staining, C-banding, silver impregnation and in situ hybridization (FISH). We were also analyze the evolutionary dynamic of the NOR-bearing chromosome in species of genus under a phylogenetic view. The results indicate that P. palliatus, P. ayeaye, and P. megacephalus have similar karyotypes, which are typical of the genus Pithecopus. In P. palliatus the NOR was detected in the pericentromeric region of pair 9p whereas in P. ayeaye and P. megacephalus we report cases of the multiple NOR sites in karyotypes. In P. ayeaye the NOR was detected in the pericentromeric region of pair 9p in both homologues and additional sites was detected in pairs 3q, 4p, and 8q, all confirmed by FISH experiments. Already in P. megacephalus the NOR sites were detected in pericentromeric region homologues of pair 8q and additionally in one chromosome of pair 13q. A comparative overview of all the Pithecopus karyotypes analyzed up to now indicates the recurrence of the NOR-bearing chromosome pairs and the position of the NORs sites on these chromosomes. We hypothesized that this feature is a result of a polymorphic condition present in the common ancestor of Pithecopus. In such case, the lineages derived from polymorphic ancestor have reached fixation independently after divergence of lineages, resulting in a high level of homoplasy observed in this marker. Our findings help to fill the gaps in the understanding of the karyotype of the genus Pithecopus and reinforce the role of the evolutionary dynamics of the rDNA genes in karyotype diversification in this group.

Androgenetic haploid Japanese pufferfish (Takifugu rubripes) induced by cold shock

Here, we explored cold-shock-induced haploid androgenesis in Japanese pufferfish (Takifugu rubripes) and analysed chromosomes by fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) in sex determination locus and microsatellite genotypes to verify all-male inheritance. We also observed early embryonic development to have an insight into the cytological mechanism of cold-shock-induced gynogenesis. The chromosome number of control group was diploid (2n = 44), while in cold-shock group haploid (1n = 22) and triploid (3n = 66) embryos were detected. Cellular DNA content flow cytometry showed that the rate of haploid induction was 90%. One signal of nucleolar organizing region (NOR) was detected by silver nitrate staining and FISH using rDNA probe in androgenetic haploids from cold-shock group. SNP analysis revealed that about half of haploid embryos examined had the G genotype indicating male-specific Y chromosome. Microsatellite genotyping showed that 26 out of 29 haploid embryos from cold-shock group exclusively inherited paternally derived genotypes. Cytological observation revealed that both second polar body and egg nucleus were located on the blastodisc surface at 60 min post fertilization in cold-shock group, suggesting simultaneous extrusion of both nuclei.

Protein metabolic changes and nucleolus organizer regions activity in the lymphocytes of neonatal calves during the development of respiratory diseases.

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.

Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

ABSTRACTNucleoli have attracted interest for their role as cellular stress sensors and as potential targets for cancer treatment. The effect of DNA double-strand breaks (DSBs) in nucleoli on rRNA transcription and nucleolar organisation appears to depend on the agent used to introduce DSBs, DSB frequency and the presence (or not) of DSBs outside the nucleoli. To address the controversy, we targeted nucleoli with carbon ions at the ion microbeam SNAKE. Localized ion irradiation with 1–100 carbon ions per point (about 0.3–30 Gy per nucleus) did not lead to overall reduced ribonucleotide incorporation in the targeted nucleolus or other nucleoli of the same cell. However, both 5-ethynyluridine incorporation and Parp1 protein levels were locally decreased at the damaged nucleolar chromatin regions marked by γH2AX, suggesting localized inhibition of rRNA transcription. This locally restricted transcriptional inhibition was not accompanied by nucleolar segregation, a structural reorganisation observed after inhibition of rRNA transcription by treatment with actinomycin D or UV irradiation. The presented data indicate that even multiple complex DSBs do not lead to a pan-nucleolar response if they affect only a subnucleolar region.

Relationships between the structural and functional organization of the turtle cell nucleolus

The nucleolus is a multifunctional structure of the eukaryotic cell nucleus. However, its primary role is ribosome formation. Although the factors and mechanisms involved in ribogenesis are well conserved in eukaryotes, two types of nucleoli have been observed under the electron microscope: a tricompartmentalized nucleolus in amniotes and a bicompartmentalized nucleolus in other species. A recent study has also revealed that turtles, although belonging to amniotes, displayed a nucleolus with bipartite organization, suggesting that this reptile group may have carried out a reversion phenomenon during evolution. In this study, we examine in great detail the functional organization of the turtle nucleolus. In liver and spleen cells cultured in vitro, we confirm that the turtle nucleolus is mainly formed by two components: a fibrillar zone surrounded by a granular zone. We further show that the fibrillar zone includes densely-contrasted strands, which are positive after silver-stained Nucleolar Organizer Region (Ag-NOR) staining and DNA labelling. We also reveal that the dense strands condensed into a very compact mass within the fibrillar zone after a treatment with actinomycin D or 5,6-dichlorobenzimidazole riboside. Finally, by using pulse-chase experiments with BrUTP, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we show that the topological and spatial dynamics of rRNA within the nucleolus extend from upstream binding factor (UBF)-positive sites in the fibrillar zone to the granular zone, without ever releasing the positive sites for the UBF. Together, these results seem to clearly indicate that the compartmentalization of the turtle nucleolus into two main components reflects a less orderly organization of ribosome formation.

A clinicopathological study and evaluation of significance of argyrophilic staining of nucleolar organizer regions in kidney tumours

A variety of benign and malignant neoplasms arise in the kidneys of adults and children. With the advancement in the recognition of newer variants and wide spectrum of appearances of these tumours especially of nephroblastoma and renal cell carcinoma, the diagnostic accuracy and assessment of critical prognostic indicators for these tumours pose challenges to the surgical pathologists. The present spurt of interest in grading the tumours on morphological and histological criteria and correlating them with AGNOR counts and to the clinical outcome in terms of survival rates is mainly attributable to the recent innovations in surgical and medical oncology. In the present study each of the renal tumour samples were subjected to an argyrophilic staining for the nucleolar organizer region according to the modified colloidal silver technique. In Grade I, II & III renal carcinoma cases the AgNOR index was 0.875, 1.301, and 1.710 respectively. It has been observed that mean AgNOR indices are directly proportional to grading. i.e., with increased grades the mean AgNOR count also increased and can be used for assessing the clinical outcome and prognosis of the cases. The mean AgNOR scores in epithelial, mesenchymal, blastemal areas for all cases of Wilms’ tumour were 4.46, 4.82, and 5.76 respectively. Based on the mean AgNOR score it has been found that blastemal areas have higher score than mesenchymal and epithelial areas. The increased score in blastemal areas can be attributed to increased proliferative activity of the tumour. Prognostication is the most important contribution of the medical scientist to the suffering patient. The AgNOR scoring as a prognostic index helps the clinician in this.

The Karyotypic Diversification of Calophysines and the Exallodontus-Propimelodus Clade (Pimelodidae, Siluriformes): A Cytotaxonomic and Evolutionary Approach in Pimelodidae Based on Ancestral State Reconstruction.

Pimelodidae family is one of the most diverse and widely distributed fish groups in South America. Phylogenetic analysis in the family have recently indicated the existence of two main clades: "sorubiminae" and the OCP clade, including Pimelodus ornatus, "calophysines" and "pimelodines." The aim of this study was to investigate the karyotype of three Amazonian Pimelodidae species: Calophysus macropterus, Propimelodus eigenmanni, and Exallodontus aguanai associating them to the literature, seeking to reconstruct probable ancestral characters. C. macropterus has 2n = 50, 20m+20sm+10a (fundamental number [FN] = 90), simple interstitial nucleolar organizing regions (NORs), and four 5S rDNA sites terminals, two in synteny with the 18S rDNA. P. eigenmanni has 2n = 56, 28m+20sm+2st+6a (FN = 106), simple NORs, and two 5S rDNA sites terminals. E. aguanai has 2n = 56, 36m+12sm+2st+6a (FN = 106) and 18S and 5S rDNA sites interstitial syntenic in the chromosome 1. All species exhibited a higher amount of heterochromatin, differing from the pattern of the family, and strong marking associated with NORs. The integration between molecular phylogenetic data and karyotype data indicated a high probability that 2n = 56 and simple terminals NORs in the short arm are ancestral characters in Pimelodidae, evidenced in "sorubiminae." In the OCP clade derived traits were observed resulting from chromosomal changes that played a critical role in the karyotype evolution of the group.

Chromosomal study of Khawia abbottinae (Cestoda: Caryophyllidea): karyotype and localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH).

An original cytogenetic study combining classical karyotype analysis and modern fluorescence in situ hybridization using telomeric (TTAGGG)n and ribosomal sequences (18S rDNA) was performed in Khawia abbottinae (Cestoda, Caryophyllidea), a parasite of Chinese false gudgeon (Abbottina rivularis) from China. Analyses based on conventional Giemsa staining, DAPI, YOYO-1 dye, and silver (Ag) staining were also carried out. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n=5m + 3t). Constitutive heterochromatin was mainly positioned at pericentromeric regions, and telomeric sequences (TTAGGG)n were restricted to the end of all chromosomes. In mitotic preparations stained with Giemsa, both homologues of chromosome pair 4 showed a distinct secondary constriction. FISH with rDNA probe confirmed that this secondary constriction contains a nucleolar organizer region (NOR). The process of spermatocyte meiosis and the dynamics of nucleolus degradation in dividing cell were scrutinized. The present study and its results enhance the limited knowledge on basic karyotype characteristics and 18S rDNA clusters location in caryophyllidean tapeworms.

Banded karyotype of Nelore cattle (Bos taurus indicus Linnaeus, 1758).

Chromosome banding techniques were applied and standardized to obtain karyotype characteristics for the first time in Brazil of Nelore cattle – Bos taurus indicus Linnaeus, 1758 – (bovine subspecies most prominent in Brazilian livestock). Blood samples were collected from the animals of the School of Agrarian and Biological Sciences of the Pontifical Catholic University of Goiás, two males and two females of pure breed. These samples were submitted to the cell culture method to study metaphase chromosomes. Chromosome banding techniques (C, G and NOR) revealed the karyotype architecture of Nelore cattle common with that of other breeds of zebu cattle formerly karyotyped. The diploid chromosome number was invariably normal, 2n = 60. C-banding revealed C-positive heterochromatin in centromeric regions almost in all chromosomes. G-banding presented the expected band pattern in the respective chromosome pairs in correspondence with the established chromosomal patterns for the species. Ag-staining for nucleolus organizer regions (AgNOR) was identified on the telomeric end of the long arm in 7 autosomal chromosomes. In this study we found more regions in chromosomes with staining than presented in the literature for the Bos indicus group (BIN). These NOR regions were repeated on the same chromosomes for the 4 animals studied.

Chromosomal Diversity of Thorny Catfishes (Siluriformes-Doradidae): A Case of Allopatric Speciation Among Wertheimerinae Species of São Francisco and Brazilian Eastern Coastal Drainages.

Wertheimerinae is a small subfamily of thorny catfish composed of two species found in eastern Brazilian coastal drainages: Wertheimeria maculata and Kalyptodoras bahiensis. According to molecular phylogenetic analysis, Franciscodoras marmoratus an endemic species of the São Francisco River is also a member of this subfamily. Even though both phylogenetic approaches suggest that this group is one of the oldest lineages of the Doradidae, a disagreement remains about the constitution of Wertheimerinae. Hence, cytogenetic analysis is important to understand the karyotypic evolution of thorny catfish and can be a useful cytotaxonomic tool to clarify the relationships between these species. All Wertheimerinae species, and F. marmoratus here analyzed, shared 2n = 58 chromosomes, karyotypic formulas (24m+12sm +8st +14a), and nucleolus organizer region (NOR) pattern (terminal 18S rDNA sites on pair 22). Differences were noted in heterochromatin and 5S rDNA site distribution. The chromosomal markers here applied added to the molecular data, reinforcing that these three species actually represent a well-resolved taxonomic unit. Our results represent one more evidence of the ancient connectivity between eastern coastal drainages and São Francisco River, whose separation represented an important event for the allopatric speciation that produced the current forms of Wertheimerinae subfamily.