Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Karyotype plasticity in crickets: Numerical, morphological, and nucleolar organizer region distribution pattern of Anurogryllus sp.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Comparison of exfoliative pap stain and AgNOR counts of the tongue in smokers and nonsmokers

Objective To compare exfoliative cytology from the oral mucosa of smokers and nonsmokers, with emphasis on proliferative activity. Methods Exfoliative cytology specimens were obtained from clinical normal mucosa from the lateral border of the tongue in 30 nonsmokers and 30 smokers ranging in age from 40 to 70 years of age, who were seen at the Heart Institute's Patient Center and the Smoking Cessation Program of the University Hospital, University of São Paulo Medical School (InCor-HCFMUSP). The cytologic specimens were evaluated by Papanicolaou staining and AgNOR quantification in order to evaluate the presence of cytological alterations suggestive of inflammation, dysplasia, keratinization, and proliferative activity of epithelial cells. Results Only Papanicolaou Class I and Class II smears were observed. Inflammatory alterations were found in 90% of smokers and in 87% of nonsmokers. The number of AgNORs/nucleus differed significantly between smokers and nonsmokers (3.372 ± 0.375 versus 2.732 ± 0.236). Conclusions Within the limitations of this research, the results indicate higher proliferative activity in smoking patients compared to nonsmoking patients, even in the absence of clinical lesions. © The Author(s) 2008.

Histologic grading and nucleolar organizer regions in oral squamous cell carcinomas

OBJECTIVE: The purposes of this study were to histologically assess different types of oral squamous cell carcinoma and the silver-binding nucleolar organizer region (AgNOR) morphology in neoplastic cells, as well as to quantify the number of AgNORs in each type of carcinoma in order to relate AgNOR count and histologic grading. MATERIAL AND METHODS: Twenty-eight cases of oral squamous cell carcinoma were divided into 4 groups, namely well-differentiated, moderately differentiated, poorly differentiated, and undifferentiated. For NOR study, 3-µm-thick sections were stained with 50% aqueous silver nitrate solution. The predominant microscopic pattern of NORs was determined. Quantitative analyses of NORs were obtained of all cells present on each histological field using a 0.025 mm² eyepiece graticule. Different histological fields were analyzed until the total number of NORs was 120 cells for each tumor. Kruskall-Wallis test was applied to compare the groups of sample data at a significance level of p=0.05. RESULTS: The mean number of AgNORs per nucleus was 3.20 for the well-differentiated group, 5.33 for the moderately differentiated one, 8.27 for the poorly differentiated one, and 10.08 for the undifferentiated one. AgNOR count was significantly different (p<0.05) among all of the studied groups. CONCLUSION: AgNOR staining technique seems to be a useful diagnostic tool since differences in AgNOR numeric values can be identified in the different types of oral squamous cell carcinoma. This technique is easy to handle and inexpensive, thus justifying its large use in histopathology.

Characterization and genome organization of a repetitive element associated with the nucleolus organizer region in leporinuselongatus (anostomidae: Characiformes)

Chromosome mapping and studies of the genomic organization of repetitive DNA sequences provide valuable insights that enhance our evolutionary and structural understanding of these sequences, as well as identifying chromosomal rearrangements and sex determination. This study investigated the occurrence and organization of repetitive DNA sequences in Leporinus elongatus using restriction enzyme digestion and the mapping of sequences by chromosomal fluorescence in situ hybridization (FISH). A 378-bp fragment with a 54.2% GC content was isolated after digestion with the SmaI restriction enzyme. BLASTN search found no similarity with previously described sequences, so this repetitive sequence was named LeSmaI. FISH experiments were conducted using L. elongatus and other Anostomidae species, i.e. L. macrocephalus,L. obtusidens, L. striatus, L. lacustris, L. friderici, Schizodon borellii, S. isognathus, and Abramites hypselonotus which detected signals that were unique to male and female L. elongatus individuals. Double-FISH using LeSmaI and 18S rDNA showed that LeSmaI was located in a nucleolus organizer region (NOR) in the male and female metaphases of L. elongatus. This report also discusses the role of repetitive DNA associated with NORs in the diversification of Anostomidae species karyotypes. Copyright © 2012 S. Karger AG, Basel.

Phyllodes tumors of the breast: Correlation of nucleolar organizer regions with histopathological malignancy grading, flow cytometric DNA analysis and clinical outcome

To examine whether nucleolar organizer regions detected by argyrophilia (Ag-NOR counts) can be used as a prognostic indicator in phyllodes tumors of the breast, and to compare its usefulness with that of DNA flow cytometric analysis, 28 cases of breast phyllodes tumors (including 15 benign, two borderline and 11 malignant tumors) were subjected to Ag-NOR staining and counting as well as DNA flow cytometric analysis. S-phase fraction and DNA ploidy analysis showed useful trends for improving outcome predictions in malignant phyllodes tumors. However, high Ag-NOR counts were significant in predicting survival status (P = 0.013) and reached near statistical significance in predicting survival times (P = 0.07). In predicting survival status, results for Ag-NOR counts were significantly better than those for ploidy analysis (P = 0.02) and S-phase fraction (P < 0.01). Only S-phase fraction was significantly predictive of survival times (P = 0.025). It is concluded that Ag-NOR counts and DNA flow cytometric analysis, easily performed using paraffin sections, give information that can improve predictions made by histopathological classification. Ag-NOR counts are significant in predicting survival in the presence of histopathological features of malignancy.

Variability of the nucleolar organizer activity in human lymphocytes via Ag-staining.

Chromosomes with Ag staining that varies from one metaphase to the other can be distinguished from those with an Ag-staining that is the same in all metaphases. The intercellular variation of an Ag-NOR can be attributed to many different factors. Whatever the importance of technical factors, they do not seem to account for the large variations in Ag-staining which were observed for each ac. This suggests the existence of a natural intercellular variability of the NOR's activity. The variation of the Ag-stainability of a given NOR, the diversity of Ag-stainings observed on the ten ac of one individual and the differences that exist between individuals raise the question of the existence of a compensation of activity between nucleolar organizers. The study, for each individual, of the mean sum of staining per metaphase reveals that this value is not absolutely constant from one individual to another; in the carriers of Robertsonian fusions it is smaller than in chromosomally normal individuals. The analysis of the transmission shows that inactive NORs remain inactive and that active NORs present a variation in the activity from one generation to the next.

Karyotype Conservation in 2 Populations of the Parthenogenetic Scorpion Tityus serrulatus (Buthidae): rDNA and Its Associated Heterochromatin Are Concentrated on Only One Chromosome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Argyrophilic nucleolar organizer regions (AgNORs) in odontogenic myxoma (OM) and ameloblastic fibroma (AF)

Ten cases of odontogenic myxoma (OM) and six cases of ameloblastic fibroma (AF) were subjected to comparative analysis by the AgNOR technique, in order to determine a possible difference in cell proliferation index between these lesions. The mean AgNOR number of the mesenchymal component of AF was compared with its epithelial component and the difference was not found to be statistically significant. The mean AgNOR index of the AF group was significantly higher than that of the OM group. Moreover, the mesenchymal component of AF demonstrated increased AgNOR numbers compared with that of OM (P<0.05). These results suggest that the epithelial and mesenchymal components of AF may have similar cell proliferative activity. However, the cell proliferative index of this lesion seems to be higher than that of OM.

Heteropicnotic chromatin and nucleolar activity in meiosis and spermiogenesis of Limnogonus aduncus (Heteroptera, Gerridae): a stained nucleolar organizing region that can serve as a model for studying chromosome behavior.

Males of Limnogonus aduncus were found to have the sex chromosome system X0 and chromosome number 2n = 23 (22A + X0). Testis cells were stained with lacto-acetic orcein and silver nitrate so that changes in the morphology and degree of staining of the heteropicnotic chromatin and the nucleolar material could be observed during meiosis and spermiogenesis. These structures share the same nuclear position and could be seen until almost the end of spermiogenesis. A chromosome region stained with silver nitrate was indicative of a nucleolar organizing region (NOR), which is rarely detected in Heteroptera with this technique. The NOR is located at one end of a single member of an autosome pair. The finding of this stained region enabled us to observe that the telomeric association of sister chromatids that characterizes the Heteroptera does not include the chromosome ends, where NORs are located; we also observed in anaphase that the chromosome end through which it is pulled to the pole is the one containing the NOR. Another observation was that the single nucleolar body present in the cells at anaphase never goes to the cell pole that does not receive the NOR. We conclude that L. aduncus is a good model for cytogenetic studies involving nucleolar activity and also may be useful for studying the mechanisms of activation and inactivation of kinetic activity at the chromosome ends. Although the chromosomes of Heteroptera are known to be holocentric, whether kinetic activity is restricted to one or involves both chromosome ends is still not well understood.

Evaluation of argyrophilic nucleolar organizer regions in oral tumor progression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Electron Tomography of Metaphase Nucleolar Organizer Regions: Evidence for a Twisted-Loop Organization

Metaphase nucleolar organizer regions (NORs), one of four types of chromosome bands, are located on human acrocentric chromosomes. They contain r-chromatin, i.e., ribosomal genes complexed with proteins such as upstream binding factor and RNA polymerase I, which are argyrophilic NOR proteins. Immunocytochemical and cytochemical labelings of these proteins were used to reveal r-chromatin in situ and to investigate its spatial organization within NORs by confocal microscopy and by electron tomography. For each labeling, confocal microscopy revealed small and large double-spotted NORs and crescent-shaped NORs. Their internal three-dimensional (3D) organization was studied by using electron tomography on specifically silver-stained NORs. The 3D reconstructions allow us to conclude that the argyrophilic NOR proteins are grouped as a fiber of 60–80 nm in diameter that constitutes either one part of a turn or two or three turns of a helix within small and large double-spotted NORs, respectively. Within crescent-shaped NORs, virtual slices reveal that the fiber constitutes several longitudinally twisted loops, grouped as two helical 250- to 300-nm coils, each centered on a nonargyrophilic axis of condensed chromatin. We propose a model of the 3D organization of r-chromatin within elongated NORs, in which loops are twisted and bent to constitute one basic chromatid coil.

Vesicle-associated proteins and transmitter release from sympathetic ganglionic boutons

A method is reported for introducing peptides derived from SNARE proteins that control exocytosis of vesicles at boutons formed by sympathetic ganglion cells in tissue culture. These peptides were coupled to the DNA binding domain of the Drosophila transcription factor antennapedia, called penetratin, This facilitated the passage of peptides across the bouton membrane. FMI-43 was used to monitor the exocytosis of transmitter from depolarized boutons after their exposure to the penetratin-peptide sequences IETRHNEIIKLETSIRELHD of syntaxin and KGFLSSLFGGSSK of alpha -SNAP. both of which blocked secretion, whereas the peptide sequences SELDDRA-DALQAGASQFETSAAKLKRK of synaptobrevin did not. This report introduces a readily applicable method for determining the effect of different peptide sequences of vesicle-associated proteins on secretion at vertebrate boutons and presents an account of the effects of a selection of such peptides on exocytosis. NeuroReport 12:607-610 (C) 2001 Lippincott Williams & Wilkins.