Active Nucleolar Organizer Regions Research Articles

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in the characid fish Triportheus venezuelensis (Characiformes, Characidae)

The karyotype and chromosomal characteristics of the characid fish Triportheus venezuelensis were investigated using differential staining techniques (C-banding, Ag-NOR staining) and fluorescent in situ hybridization (FISH) with an 18S rDNA probe. The diploid chromosome number (2n = 52), karyotype composition and sex chromosome determination system of the ZZ/ZW type were the same as previously described in other species of the genus Triportheus. However, extensive variation regarding nucleolus organizer regions (NOR) different from other species was observed. 18S rDNA sequences were distributed on nine chromosome pairs, but the number of chromosomes with Ag-NORs was usually lower, reaching a maximum of four chromosomes. When sequential staining experiments were performed, it was demonstrated that: 1.) active NORs usually corresponded to segments with 18S rDNA genes identified in FISH experiments; 2.) several 18S rDNA sequences were not silver-stained, suggesting that they do not correspond to active NORs; and 3.) some chromosomes with silver-stained regions did not display any 18S rDNA signals. These findings characterize an extensive polymorphism associated with the NOR-bearing chromosomes of T. venezuelensis and emphasize the importance of combining traditional and molecular techniques in chromosome studies.

Efficient repair of bleomycin-induced double-strand breaks in barley ribosomal genes

Ability of barley ribosomal genes to cope with damage produced in vivo by the radiomimetic agent bleomycin was investigated. Repair kinetics of bleomycin-induced double-strand breaks in ribosomal and total genomic DNA was compared. Induction and repair of double-strand breaks in defined regions of the ribosomal genes was also analyzed. Preferential sensitivity of barley linker DNA towards bleomycin treatment in vivo was established. Relatively higher yield of initially induced double-strand breaks in genomic DNA in comparison to ribosomal DNA was also found. Fragments containing intergenic spacers of barley rRNA genes displayed higher sensitivity to bleomycin than the coding sequences. No heterogeneity in the repair of DSB between transcribed and non-transcribed regions of ribosomal genes was detected. Data indicate that DSB repair in barley rDNA, although more efficient than in genomic DNA, does not corelate with the activity of nucleolus organizer regions.

Nucleolar dominance: a model for rRNA gene silencing

Research over the last decade has demonstrated that the regulated packaging of DNA into chromatin is fundamental to keeping genes in an active/open or a more condensed/inactive conformation. Nucleosomes, the basic repeating unit of chromatin, contain two molecules each of canonical histones, H2A, H2B, H3, and H4. These are organized as an H3–H4 tetramer associated with two H2A–H2B dimers. The NH2 termini of histones protrude from the nucleosome, and are the target for a wide array of covalent modifications including acetylation, methylation, and phosphorylation. These modifications are applied and removed in a highly specific manner to generate what has been described as the histone code (Jenuwein and Allis 2001). This code is, in turn, read by chromatin-associated factors. For example, individual acetylated lysine residues are recognized by factors with bromodomains and methylated lysines by chromodomain-containing proteins. Additionally, the action of ATP-dependent chromatin remodeling activities can displace nucleosomes altering the accessibility of DNA within chromatin templates (Flaus and OwenHughes 2001). While most of this work has focused on protein-encoding genes transcribed by RNA polymerase II, more recent studies, including one by Earley et al. (2006) in this issue of Genes & Development, are aimed at elucidating the role that chromatin plays in determining the activity status of the genes that encode ribosomal RNAs (rRNAs). Eukaryotic genomes contain many rRNA gene copies, ranging from hundreds to thousands in some plants, organized in tandem arrays. rRNA genes are transcribed by RNA polymerase I (Pol I) into a precursor RNA (prerRNA) that encodes the three largest RNA components of ribosomes. Pre-rRNA coding sequences are separated by intergenic spacers (IGS) that can range in size from 3 kb in yeast to 30 kb in mammals. Pre-rRNA synthesis is regulated by elements including promoters, transcriptional enhancers, and terminators that are located within the IGS (Grummt 1999). Transcription of rRNA gene arrays results in formation of a nucleolus; consequently, they are termed nucleolar organizer regions (NORs). Importantly, not all NORs are transcriptionally active. Species with multiple NORs can vary the active proportion. Pre-rRNA is matured into 18S, 5.8S, and 28S rRNA by a precisely ordered series of events that includes both cleavages and targeted base modifications. These are required to ensure correct folding of rRNAs for ribosome assembly and function. These multiple steps in ribosome biogenesis require a bewildering array of proteins and small nucleolar RNAs that converge on transcriptionally active NORs, forming such a high concentration of ribonucleoprotein complexes that nucleoli are the most prominent feature of the eukaryotic nucleus. The electron micrographs of Miller and Beatty (1969) demonstrated that the coding regions of active repeats are fully loaded with Pol I. rRNA gene transcription accounts for ∼50% of nascent RNA synthesis in a cell. This high density of transcription and the recent demonstration that it is tightly coupled to pre-rRNA processing (Granneman and Baserga 2004) argues that rRNA gene chromatin needs to be especially accessible. As cells approach metaphase, Pol I transcription is actively shut down and the nucleolus disappears. It is at this point that the specialized nature of active rRNA gene chromatin is most dramatically revealed by the appearance of NORs as secondary constrictions on metaphase chromosomes (McClintock 1934).

Cytogenetics of two sympatric Corydoras species (pisces, siluriformes, challichtyidae) of Southern Brazil

Karyotypic data are presented for two sympatric Corydoras species of the Lagoa Dourada, namely, C. ehrhadti and C. paleatus, which are found in the upper Tibagi river basin (Ponta Grossa, State of Paraná, Brazil). The same diploid number and karyotypic formula were observed in both species/populations. A great similarity in the constitutive heterochromatin distribution and in the activity of nucleolar organizer regions was also found. The use of in situ hybridization with a fluorescent 18S rDNA probe allowed for the identification of the species/populations through the location of ribosomal sites.

Cytogenetic divergence between two sympatric species of Characidium (Teleostei, Characiformes, Crenuchidae) from the Machado River, Minas Gerais, Brazil

Cytogenetic studies were performed on two sympatric species of Characidium, C. gomesi and C. cf. zebra, from the Grande River basin, Minas Gerais State, Brazil. Although both species had a chromosome number of 50 with a karyotype exclusively consisting of meta- and submetacentric chromosomes, interspecific diversity was detected concerning the size of the two first chromosome pairs of the karyotypes. Active nucleolus organizer regions (NORs) were located at the terminal position on the long arm of the 17th pair of C. gomesi and at subterminal position on the long arm of the 23rd pair of C. cf. zebra. For both species the fluorochrome CMA3 stained only the NOR-bearing pair of chromosomes. The heterochromatin pattern also showed some differentiation between these species restricted to the centromeric or pericentromeric region of C. cf. zebra and practically absent in C. gomesi. These data are discussed concerning chromosome diversification in this fish group.

A role for upstream binding factor in organizing ribosomal gene chromatin

Human ribosomal genes are located in NORs (nucleolar organizer regions) on the short arms of acrocentric chromosomes. During metaphase, previously active NORs appear as prominent chromosomal features termed secondary constrictions, which are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I transcription factor UBF (upstream binding factor) binds extensively across the ribosomal gene repeat throughout the cell cycle. Evidence that UBF underpins NOR structure is provided by an examination of cell lines in which large arrays of a heterologous UBF binding sequences are integrated at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus, and during metaphase form novel silver-stainable secondary constrictions, termed pseudo-NORs, that are morphologically similar to NORs.

Nucleolar organizer activity and competition in Tricepiro Don René INTA, a synthetic forage crop

The nucleolar organizer activity and the presence of rDNA zones were studied in Tricepiro Don Rene ´ INTA, a synthetic forage crop (2n=42, 6x, AABBRR with introgression of Thinopyrum in 6A chromosome). The use of pTa71 probe revealed the presence of six rDNA zones but Ag-NOR indicated the presence of only four NORs (nucleolus organizer regions). Differential amphiplasty phenomenon could explain the inactivity of some rDNA zones. Simultaneous use of FISH (pTa71 probe) and DAPI banding analysis indicated that the chromosomes with rDNA zones belong to two rye and to four wheat chromosomes. The maximum number of nucleoli observed in so- matic cells and the number of chromosomes with secondary constrictions agreed with the four active NORs detected with the Ag-staining method. The use of the pSc119.2 probe allowed to recognize that chromosomes with secondary constrictions are the wheat chromosomes 1B and 6 B. The results obtained using classical and molecular cytogenetics lead us to conclude that this crop has a similar be- havior to that observed in hexaploid triticales in relation to the phenomenon of amphiplasty. Furthermore, the com- plexity of genetic interactions between wheat and rye genomes described in triticales, are not modified by the pres- ence of Thinopyron introgression in Tricepiro Don ReneINTA.

NOR expression increases on metaphase chromosomes of down syndrome lymphocytes in concordance with mitogen concentration in culture medium

Regulation of nucleolus organizer region (NOR) expression in trisomy 21 (Down syndrome [DS]) cells is not fully explained. This work compared NOR expression on metaphase chromosomes in gradiently stimulated lymphocytes from DS patients with those from healthy controls. Conventional peripheral blood culture (72 h) and chromosomal preparation procedures were used except that blood samples from each individual were cultivated in the same but gradiently increasing concentrations (0.37, 0.75, 1.48, and 2.21 ml) of phytohemagglutinin (PHA) per 100 ml of medium. One hundred consecutive metaphases per concentration were analyzed for scoring the means of the active NORs bearing chromosomes (AgNOR+ chromosome) per individual and per concentration. In contrast to healthy controls (n=24), AgNOR+ chromosomal number in lymphocytes from 30 DS patients increased in concordance to the gradient of PHA concentration in the culture medium. DS lymphocytes do not downregulate their NOR expression in the limit of control cells. This in vitro result may serve as a clue for the explanation of the DS phenotype due to the wasted energy in producing unnecessary rRNA transcripts and AgNOR proteins in utero during organogenesis. These results also indicate that precautions must be used in routine work of NOR evaluation/interpretation in DS lymphocytes.

AgNOR variability among Robertsonian races of the house mouse from the island of Madeira: implications for patterns of Rb fusion formation and genetic differentiation

The island of Madeira harbours highly differentiated Robertsonian (Rb) races of the house mouse (Mus musculus domesticus), carrying several unique Rb fusions. The reproductive and geographical isolation between these races is expected to restrict gene flow severely, and contribute to their genetic differentiation. In the present study, the chromosomal distribution and frequency of transcriptionally active nucleolar organizer regions (AgNORs) between five Madeiran races was studied. The results showed that AgNORs were present in the centromeric regions of five chromosome pairs. Differences in NOR distribution were apparent within and between races. Comparison between races showed that AgNOR variation occurred independently of the Rb or non-Rb state of the chromosomes and of the NOR-bearing status of the partner chromosomes. The results indicated that Rb fusion formation does not interfere with the activity of rDNA genes. In addition our data provided no evidence that the presence of AgNOR sites inhibits Rb fusion. A cluster analysis showed that AgNOR differentiation was only partly congruent with the chromosomal divergence and geographical proximity between races. In particular, parapatrically distributed races showing the lowest chromosomal divergence exhibited high levels of AgNOR differentiation. If confirmed, this suggests that premating isolation may be reinforcing a barrier to gene flow between the races.

Cytogenetic analysis of platyfish (Xiphophorus maculatus) shows location of major and minor rDNA on chromosomes

Chromosome distribution of nucleolus organizer regions (NORs) and 5S rDNA loci were investigated in platyfish (Xiphophorus maculates) using conventional and molecular cytogenetic techniques. Chromomycin A3 (CMA3) staining of GC- rich heterochromatin provided four CMA3 cytotypes in studied platyfish. As in fish, CMA3 positive sites are related to NORs, chromosomes sequentially stained by CMA3 and AgNO3 showed bright blocks that were transcriptionaly active NORs. Additionally, PRINS with primers enabling amplification of 5S rDNA and CMA3 staining revealed existence of 5S rDNA sites located close to GC rich regions. The analysis could suggests that minor and major rDNAs may be linked.

UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery.

Human ribosomal genes (rDNA) are located in nucleolar organizer regions (NORs) on the short arms of acrocentric chromosomes. Metaphase NORs that were transcriptionally active in the previous cell cycle appear as prominent chromosomal features termed secondary constrictions that are achromatic in chromosome banding and positive in silver staining. The architectural RNA polymerase I (pol I) transcription factor UBF binds extensively across rDNA throughout the cell cycle. To determine if UBF binding underpins NOR structure, we integrated large arrays of heterologous UBF-binding sequences at ectopic sites on human chromosomes. These arrays efficiently recruit UBF even to sites outside the nucleolus and, during metaphase, form novel silver stainable secondary constrictions, termed pseudo-NORs, morphologically similar to NORs. We demonstrate for the first time that in addition to UBF the other components of the pol I machinery are found associated with sequences across the entire human rDNA repeat. Remarkably, a significant fraction of these same pol I factors are sequestered by pseudo-NORs independent of both transcription and nucleoli. Because of the heterologous nature of the sequence employed, we infer that sequestration is mediated primarily by protein-protein interactions with UBF. These results suggest that extensive binding of UBF is responsible for formation and maintenance of the secondary constriction at active NORs. Furthermore, we propose that UBF mediates recruitment of the pol I machinery to nucleoli independently of promoter elements.

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.

The odd-even effect in mitotically unstable B chromosomes in grasshoppers

The odd-even effect, by which B chromosomes are more detrimental in odd numbers, has been reported in plants and animals. In grasshoppers, there are only a few reports of this effect and all were referred to as traits related to the formation of aberrant meiotic products (AMPs). Here we review the existing information about B chromosome effects on AMPs, chiasma frequency and the number of active nucleolus organizer regions (NORs) per cell. Polysomy for A chromosomes and B chromosomes are two kinds of chromosome polymorphism frequently found in grasshoppers. In some aspects, e.g. meiotic behaviour and mitotic instability leading to individual mosaicism (in the case of mitotically unstable Bs), polysomic As show similar characteristics to B chromosomes. In fact, polysomy is regarded as one of the main mechanisms for B chromosome origin. Here we review some features of meiotic behaviour in known cases of polysomy and mitotically unstable Bs in grasshoppers, in looking for possible causes for the odd-even effect. In all these traits, the odd-even effect was apparent, although its appearance was not universal in any case, with variation among species or populations within the same species. The equational division and lagging of the extra chromosomes, when univalents, could favour the appearance of abnormal meiotic products, and the formation of bivalents, when there are two or more extra chromosomes, inhibits this process. Therefore, the odd-even effect might be a consequence of the concomitant operation of both aspects of extra chromosome meiotic behaviour. The possibility that the odd-even effect might result from an increase in cell stress generated by odd numbers is suggested.

Variation in size of Ag-NORs and fluorescent rDNA in situ hybridization signals in six breeds of domestic pig.

Variation of the size of silver-stained nucleolar organizer regions (NORs) of chromosomes 10 and 8 was studied in pigs of six breeds (Sus scrofa L.). The silver deposits were quantified by image analysis and the results were normalized for each Ag-NOR chromosome. In general, normalized values for chromosomes 10 were higher than those for chromosomes 8, suggesting that the NOR activity of chromosomes 10 is dominant as compared to that of chromosomes 8. However, high values for chromosomes 8 were found in the Meishan breed and in some Piétrain pigs, indicating a high transcriptional activity of the rRNA genes on these chromosomes. In some pigs, the relative quantities of rDNA in chromosomes 10 and 8 were investigated by fluorescent in situ hybridization and the results were compared with those of the silver staining procedure. It is concluded that Ag-NOR sizes on chromosomes 10 are relatively well correlated to the number of rRNA genes, whereas the absence or the small size of Ag-NORs on chromosomes 8, often observed in pigs, is the result of low NOR activity rather than of absence of rDNA.

A New Method for Differential Staining of Brassica Metaphase Chromosomes, and Karyotypes of B. Campestris, B. Oleracea, and B. Napus

Mitotic cells from three Brassica species, B. campestris L., B. oleracea L., and B. napus L. have been differentially stained after cold treatment and application of the ASG method for G-banding. Karyotypes and idiograms are presented. In all three species the chromosomes can be matched to individual pairs. The heterochromatic satellite next to the nucleolar organizer region (NOR) differs from the rest of the chromosomal material by having a very faint colour. It responds to the staining procedure in the same way as cold sensitive regions of chromatin described in other species. In Brassica this type of reaction is apparently limited to the satellite next to the active NOR.

Abnormal features of Macoma balthica (Bivalvia) in the Baltic Sea: alerting symptoms of environmental adversity?

Recent studies of the Baltic clam Macoma balthica (L.) from the southern Baltic (the Gulf of Gdansk) have revealed striking morphological, histological and cytogenetic features. Strong deformation of the shell, including elongation of the posterior end and the appearance of an easily visible flexure in this part, has been recorded. The population contribution of the deformed blunt shelled (“irregular”) clams ranged from 0% to 65% and tended to increase with depth. The morphologically “irregular” clams had higher accumulated tissue concentrations of trace metals (As, Ag, Cd, Pb, Cu and Zn), indicating a different metal handling ability. Adverse conditions in deeper water regions of the Gulf (e.g. hypoxia, hydrogen sulphide, elevated bioavailability of contaminants) have been suggested as inducers of the phenotypical changes (morphological deformation) in part of the population and, in parallel, of the specific physiological adaptations that result in higher metal accumulation in the “irregular” clams. Cytogenetic and histological analyses showed the presence of tumours in gill cells and digestive system of the affected clams, the prevalence of disseminated neoplasia ranging from 0% to 94% depending on the site. The disease was manifested by a modified karyotype (i.e. an abnormal number and morphology of chromosomes), a higher activity of nucleolar organizer regions (AgNORs), and tissue lesions (enlarged cells, actively proliferative with pleomorphic nuclei). Bottom sediments showed acute toxicity and have been proposed as a source of an initialising carcinogenic factor. However, none of the ecotoxicological studies provided was successful in the clear demonstration of a single (or multifactorial) agent that can account for the disseminated neoplasia.

Use of multiple cytometric markers improves discrimination between benign and malignant melanocytic lesions: a study of DNA microdensitometry, karyometry, argyrophilic staining of nucleolar organizer regions and MIB1-Ki67 immunoreactivity.

Confident separation of benign naevi and malignant melanoma can sometimes be very difficult using conventional microscopy. This study evaluated the combined diagnostic abilities of multiple cytometric markers in separating various types of naevi from melanomas. The lesions studied included 27 benign compound naevi, 20 dysplastic naevi, 10 Spitz naevi and 24 melanomas. The cytometric features investigated were: (i) nuclear DNA content and chromatin compactness, measured by video imaged DNA microdensitometry; (ii) nuclear morphology, measured by nuclear morphometry (karyometry); (iii) transcriptional activity of nucleolar organizer regions, measured as the number and size of argyrophilic staining of nucleolar organizer regions (AgNORs); and (iv) cellular proliferative activity detected by quantifying the immunoreactivity of MIB1-Ki67 antigen. These variables were evaluated in the superficial, middle and deep zones of each lesion. Using multivariate discriminant analysis, a total diagnostic effectiveness of 97% could be achieved in separating the benign and malignant melanocytic lesions by co-evaluating variables for DNA microdensitometry, karyometry and AgNORs. A diagnostic effectiveness of 100% could be achieved if further co-evaluation with MIB1-Ki67 immunoreactivity was performed. Our study suggests that co-evaluation of multiple cytometric markers can improve the diagnostic abilities of individual techniques in separating benign naevi from malignant melanomas. This may be of particular significance in the diagnosis of melanocytic lesions whose biological behaviour cannot be confidently predicted by their histological features using conventional microscopy.

Species status of two populations of Encarsia sophia (Girault & Dodd) (Hymenoptera: Aphelinidae) native to different geographic areas

Abstract Encarsia sophia is a cosmopolitan parasitoid of whitefly pests and is regarded as a potential biocontrol agent of the Bemisia tabaci species-complex. However, the possibility exists that under this specific name a complex of cryptic species may be contained, whose correct identification is essential for their effective use in biological control programmes. In this paper two populations native to Pakistan and Spain, respectively, were characterized by means of morphometric analysis, crossing experiments and karyological analysis. The two populations, indistinguishable on the basis of single morphological characters, were distinguished through a canonical discriminant function analysis. Mating tests showed rare copulation attempts and no transfer of spermatozoa into the spermatheca in heterogamic crosses, thus suggesting that the Pakistani and Spanish populations are reproductively isolated. Although the two populations shared the same karyotype formula (2n=2m+2sm+6a), consistent differences were found in their chromosome sets. In the Spanish population the metacentric chromosome was almost one half of the whole genome set (rl=44.23), while in the Pakistani one, it was shorter (rl=29.55) and as long as the sub-metacentric chromosome. Moreover, silver staining provided evidence of a different localization of the active nucleolus organizer regions (NORs). Results obtained in this study suggest that the Pakistani and Spanish populations are distinct cryptic species.

Natural variation in nucleolar dominance reveals the relationship between nucleolus organizer chromatin topology and rRNA gene transcription in Arabidopsis.

In genetic hybrids, nucleolus formation on chromosomes inherited from only one parent is the epigenetic phenomenon, nucleolar dominance. By using Arabidopsis suecica, the allotetraploid hybrid of Arabidopsis thaliana and Arabidopsis arenosa, natural variation in nucleolar dominance was found to occur, providing a unique opportunity to examine homologous nucleolus organizer regions (NORs) in their active and inactive states. In A. suecica strain LC1, NORs derived from A. arenosa are active, whereas A. thaliana-derived NORs are silenced. In A. suecica strain 9502, NORs of both parental species are active. When active, NORs are partially, but not fully, decondensed. Both active and inactive LC1 NORs colocalize with the nucleolus, contradicting the long-standing assumption that rRNA gene transcription drives nucleolus association. Collectively, these observations clarify the relationships among NOR chromatin topology, rRNA gene transcription, and NOR-nucleolus associations. A. suecica strains LC1 and 9502 have each lost one pair of A. thaliana NORs during evolution, and amplified fragment-length polymorphism analysis further indicates that these strains are genetically very similar. These data suggest that nucleolar dominance can result from subtle genetic or epigenetic variation but is not a trait fundamental to a given interspecies hybrid combination.

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera, Culicidae) from the Brazilian Amazon

Fluorescence in situ hybridization of Anopheles darlingi and A. nuneztovari demonstrated nucleolar organizer region activity at the end of the fourth larval instar, when the nucleolar organizer regions underwent gradual condensation. The heteromorphic sex chromosomes showed intraindividual size variation in the rDNA blocks located in the pericentromeric region and this coincided with the location of constitutive heterochromatin (C-banding).