Active Nucleolar Organizer Regions Research Articles

Chromosome variability in European vendace (Coregonus albulaL.) from Poland

SUMMARYA population of European vendace, Coregonus albula from Lake Ukiel, Olsztyn District, Poland, has been karyotyped using conventional staining and banding techniques. Diploid chromosome number was 80 (NF = 96). The chromosome set consisted of 7 pairs of metacentric, 1 pair of submetacentric, 2 pairs of subtelocentric and 30 pairs of acrocentric chromosomes, gradually decreasing in size. Variation in size of one arm of second and fifth chromosome pair was observed. The presence of constitutive heterochromatin on these arms was confirmed by C-banding. The polymorphism of metacentric pairs observed in European vendace was probably due to deletion or to an involvement of heterochromatic arm in an unequal crossover. Examined fish possessed from 0 to 2 minute supernumerary chromosomes which were entirely heterochromatic, as was revealed by C-banding. The number and locations of active nucleolar organizer region (NOR) were demonstrated.

Effect of abscisic acid, gibberellic acid, indoleacetic acid, and kinetin on selective ribosomal cistron regulation in quiescent and senescent onion leaf base tissue

Small pieces of tissue from the basal, equatorial, near-apical, and apical regions of the third turgid onion leaf base were treated (3 and 6 h in the dark) with abscisic acid (ABA), gibberellic acid (GA 3), indoleacetic acid (IAA), and kinetin (K) and compared with responses in water controls. ABA inhibited the activation (increase in size and changes in morphologies from round or oval to elongated-oval and dumbbell) of major nucleolar organizer regions (NORs) in basal, equatorial, and near-apical tissue. GA 3 and K activated the major NORs in the basal, equatorial, and near-apical tissue. IAA stimulated the activation of major NORs in basal tissue but inhibited their activation in equatorial and near-apical tissue. No major nucleoli were activated in control or plant growth regulator-treated apical tissue. Minor NORs were not expressed in the control and plant growth regulator-treated tissue in these four locations. Actinomycin D and cycloheximide inhibited major NOR activation in equatorial control and kinetin-treated tissue. We propose that ABA, GA 3, IAA, and K are major NOR regulators. We infer that the basal through near-apical cells were quiescent during post-harvest storage and that the cells in the apical tissue had senesced beyond the point of no return (degeneration of the karyoskeleton) in the cellular senescence pathway.

Amplification of (GACA)n simple repeats in an exceptional 14p+ marker chromosome

An inherited 14p+ marker chromosome with an unusually large differentially staining region (DSR) on the short arm was examined with a number of banding techniques and by non-radioactive in situ hybridization using various repetitive DNA probes. The increase in the size of this variant chromosome was 40% that of a normal chromosome 14. The extra chromosomal material in the DSR consisted mainly of GC-rich constitutive heterochromatin within which two equally sized clusters of 18S + 28S ribosomal RNA genes were located. In situ hybridization demonstrated that the DNA in the DSR was highly enriched in simple tetrameric (GACA)n sequences, whereas the centromeric alphoid sequences and the (TTAGGG)n telomeric repeats were not amplified. Silver staining of the two nucleolus organizer regions (NORs) within the DSR showed that the telomerically located NOR was always more active than the paracentromerically located NOR. A comparison with other DSRs found in human acrocentric autosomes revealed a gradient of transcriptional activity of adjacent multiple NORs. This gradient decreased in the order of their telomeric-paracentromeric-interstitial position, regardless on which acrocentric chromosome the DSR was located.

Possible origin of a B chromosome deduced from its DNA composition using double FISH technique

Double fluorescent in situ hybridization (FISH) with two DNA probes (a 180 bp tandemly repeated DNA and ribosomal DNA) was performed in embryo cells of the grasshopper Eyprepocnemis plorans. Repetitive DNA was present in most standard chromosomes (excepting 7, 8 and 10) and in the proximal two-thirds of the B chromosome, which was its major location in the complement. Ribosomal DNA was present distally on the B, and in the active nucleolar organizer regions (NORs) of the X, 9, 10 and 11 chromosomes. A small number of rRNA gene clusters was also observed in the pericentromeric regions of chromosomes 1-8. The double FISH technique showed that the B chromosome (B2 type) is mainly composed of a 180 bp tandem repeat and ribosomal DNA, the minute short arm being the only region that does not hybridize with them. The location and order of the centromere and both the DNA sequences on the B chromosome coincide only with those in the X chromosome, indicating that the B most probably derives from the X.

NOR activity in larval and juvenile mussels (Mytilus gallopro vincialis Lmk.)

The silver staining patterns of the nucleolus organizer regions (NORs), an indication of rDNA transcriptional activity, were studied in metaphase chromosomes from larvae and juveniles of a bivalve mussel, Mytilus galloprovincialis. Two submetacentric/subtelocentric pairs of chromosomes, out of the the total complement of 14 pairs, were characterized by the presence of silver-stained NORs. A considerable degree of inter- and intraindividual variation in NOR activity was noted. Mean NOR activity in larvae (3.27 active NORs per metaphase) was demonstrated to be higher than in the gill tissue of the juvenile mussels (1.93).

PCNA expression and nucleolar organizer regions in malignant melanoma and nevus cell nevus.

The histogenesis of malignant melanoma with particular reference to the role of melanocytic nevus is still controversial in oncological pathology. In order to differentiate between the proliferative activity of malignant melanomas and benign melanocytic tumors, immunohistochemical analysis was carried out. We used monoclonal antibody PC10 to identify the Proliferating Cell Nuclear Antigen (PCNA), a highly conserved 36 KD acidic nuclear protein which correlates with cell proliferation, and the AgNOR method to stain the Nucleolar Organizer Regions (NORs) whose number and configurations may reflect protein synthesis activity. 47 cases of primary melanoma, 63 metastatic melanoma lesions, and 23 cases of nevi are included in this study. Spitz nevi showed a higher index of cell proliferation compared with other common benign nevi but a much lower index compared with malignant melanoma cells. The metastatic lesions of malignant melanomas had a higher index of proliferation and activity of cells than the primary lesions. Skin-metastatic lesions had higher indexes of cell proliferation and NOR activity than lymph node-metastatic lesions. These results support the idea that metastatic melanoma cells are derived from a more advanced stage of tumor progression. These data suggest that the combination of PCNA and AgNOR is an useful marker for differentiating benign melanocytic tumors from malignant melanomas.

Cytogenetic analysis of a benzpyrene induced osteosarcoma in the rat ( Rattus norvegicus)

A cytogenetic comparison of primary and transplant tumor cell-lines, both originating from a benzpyrene induced osteosarcoma, with normal rat cell-lines ( Rattus norvegicus) is presented here. In all tumor cell-lines tested, the number of chromosomes was increased by one or two. Using Giemsa-banding, structural chromosomal changes, i.e. a Robertsonian translocation t(4;4)(q10;q10) and an interstitial deletion del(6)(q11q16) could be recorded. Furthermore, staining of nucleolus organizer regions (NORs) revealed a shift in NOR activity from chromosome number 11 to 12 and a decrease in NOR activity at chromosome number 3.

Ethylene is a selective ribosomal cistron regulator in Allium cepa epidermal cells

In Allium cepa L. (onion) the number, size, and morphology of visible nucleoli per nucleus varies during cell division, growth, differentiation, storage, activation of quiescent tissue, senescence, wounding, and disease (host-pathogen interactions). Since there are two types (differentiated visually by size) of ribosomal cistron in onion (major and minor nucleoli differ in the external spacer nucleotide sequences), we inferred that they function separately. In controls, major nucleolar organizer regions (NORs) were visible in epidermal cell nuclei as nucleoli. After exposure of the tissue to ambient conditions, these nucleoli enlarged and changed morphology. Minor NORs (when in the genome) required more than 6 h to become developed as visible nucleoli. In the ethylene treatments, the major NORs (visible nucleoli in quiescent epidermal cells) increased in size (activation of a greater number of tandem rRNA genes) as in the controls. The minor, quiescent NORs became active and visible (activation of quiescent, inactive NORs) within 3 h. Actinomycin D, and cycloheximide, with or without ethylene, inhibited the increase in size of the major NORs, and prevented activation of the minor NORs (i.e. they did not become visible). Silver nitrate and cobalt chloride had no effect on major NORs but inhibited the appearance of minor nucleoli when combined with ethylene treatments. We infer: ethylene acts to regulate the expression of the minor NORs (selective ribosomal cistron regulation); and, other hormone(s) are involved in the regulation of the major NORs. Yellow and white sweet Spanish onions we studied had two major NORs and two minor NORs. Red sweet Spanish onions we studied had only the two major NORs.

Nucleolar Organizer Regions as Markers of Incipient Cellular Alterations in Squamous Epithelium

Transcriptionally active nucleolar organizer regions identified by silver staining (AgNOR) vary in number with cellular activity and/or malignant transformation and have been used as a diagnostic tool. A morphometric study of AgNORs was performed in an experimental model of irradiated squamous epithelium (Wistar rat sole skin) 4, 8, and 14 hours and 1, 2, 5, and 7 days post-irradiation with 50 Gy of x-rays. A statistically significant and progressive rise in AgNOR average volume of up to 238% and reduction in AgNOR number/nucleus of up to 40% were detected as a function of post-irradiation time. A statistically significant 46% increase in AgNOR volume was detected as early as 8 h post-irradiation, when no histological changes were observable in routine preparations. These results suggest that AgNORs may be useful as a quantitative marker of incipient changes in cellular activity and caution against the indiscriminate use of AgNORs in the follow-up of lesions which may have been exposed to radiotherapy. Furthermore, this study suggests the possibility of using AgNORs as a sensitive biological dosimeter in cases of uncontrolled exposure to radiation.

Molecular cytogenetic characterization of three familial cases of satellited Y chromosomes

Three families in which a satellited Y chromosome (Yqs) was segregating without apparent phenotypic effect were re-investigated with non-isotopic in situ hybridization methods. Active nucleolus organizer regions were seen in the distal long arm region of all Yqs chromosomes studied and in situ hybridization with the probe D15Z1 showed that, in all three families, the Yqs was the result of a 15p;Yq translocation. In one case, an additional focus of D15Z1 hybridization was seen on 21p.

Morphological changes of the nucleolar organizer regions induced by 7,12-dimethylbenz[a]anthracene in the hamster cheek pouch.

The staining of nucleolar organizer regions (NORs), which equate to rDNA transcription, was applied to chemically induced-lesions of the hamster cheek pouch. The cheek pouches of 16 male golden Syrian hamsters were treated three times a week with 0.5% 7,12-dimethylbenz[a]anthracene (DMBA) in mineral oil for 16 weeks. The percentage of gathered-type NORs with high activity nucleoli increased in the pouch epithelium during DMBA treatment and reached the highest values in malignant tumors. The percentage of dispersed-type NORs also increased in the malignant lesions. However, the absolute number of NORs was not affected by DMBA treatment. These results suggest that DMBA induces modification of NOR activity at the early stages of carcinogenesis and shows the potential of this model for studying NOR alterations in neoplasia.

High incidence of ectopic nucleolar organizer regions in human testicular tumors

Eight primary testicular germ cell tumors, one teratocarcinoma cell line, and one Leydig cell tumor were studied to determine the importance of modifications of the nucleolar organizer regions (NORs) in human testicular tumors. Cytogenetic analysis after silver staining showed active ectopic NORs in two primary embryonal carcinomas (EC) in the cell line and in single cells of each of two seminomas (S). In one EC, an ectopic NOR was localized to chromosomal region 1q4; the others were on unidentified rearranged chromosomes. All tumors in which ectopic NORs were observed were hyperdiploid and possessed marker chromosomes typical of human germ cell tumors. Quantitative DNA analysis was performed on three tumors: a teratocarcinoma (TC) and the Leydig cell tumor, which had provided no analyzable mitoses, and a seminoma which was cytogenetically diploid. In all three cases, the major populations were hyperdiploid. The results, in combination with those of an earlier study, provide evidence that active ectopic NORs are common in human testicular tumors.

Characterization of the karyotype of the tench (Tinca tinca L.) and analysis of its chromosomal heterochromatic regions by C-banding, Ag-staining, and restriction endonuclease banding.

Beginning with a description of the conventional Giemsa-stained karyotype of the tench (Tinca tinca L.), the structure and variability of the chromosomal heterochromatic regions in this cyprinid species were analyzed by means of C-, silver-, and restriction endonuclease banding. Silver staining revealed active nucleolus organizer regions (NORs) on the secondary constriction of chromosome pair 3. Constitutive heterochromatin was associated with NOR regions detected by C-banding. Restriction endonuclease digestion with AluI, TaqI, and HaeIII induced specific banding patterns that allowed identification of homologous chromosome pairs and revealed features about the sequence composition of several chromosomal heterochromatic regions and of the NOR-associated heterochromatin.

Induction of G- and R-banding in human chromosomes by the demethylating agent S-adenosyl-L-homocysteine.

In this report we describe the procedure of growing human lymphocytes with the demethylating agent S-adenosyl-L-homocysteine (SAH). After this treatment, which is not toxic for cell survival, both R- and G-banding were obtained by new experimental procedures: R-bands have been directly demonstrated with the GC-specific fluorochrome chromomycin A3 without the necessity of any AT-specific counterstaining agent; simultaneous G-banding and active nucleolar organizer regions have been obtained by silver impregnation of chromosomes and subsequent Giemsa staining. These results suggest a possible relationship between local differences in DNA methylation and the determination of the banded chromosome structure.

Increased rDNA Transcriptional Activity in Celiac Disease

The activity of nucleolar organizer regions (NORs) in chromosomes of peripheral blood lymphocyte cultures from 20 healthy subjects and 32 patients with celiac disease (CD) (nine untreated patients, nine treated but with dietary lapses, and 14 treated with gluten-free diet (GFD) and normal small bowel histology) was studied. Furthermore, three female patients diagnosed with small bowel non-Hodgkin's lymphoma (NHL) complicating CD were studied. Silver (Ag)-staining technique was used to visualize positive NORs. In each individual, 20 metaphases were analyzed to determine the number of NORs per cell. The average of Ag-NOR+ per cell, expressed as mean +/- SD, was found to be higher in the CD group (6.62 +/- 0.65) compared with controls (5.70 +/- 0.81) (p less than 0.001). This increase was evident in both groups of chromosomes analyzed (D and G). No differences were found among the three groups, but all of them were found to be statistically different compared to controls (p less than 0.001). The NHL complicating CD patients showed a statistically increased frequency of Ag-NORs (7.20 +/- 0.39) with respect to CD patients (p less than 0.02) and controls (p less than 0.001). These findings show an increase of the transcriptional activity of rDNA in CD that could be related to the high incidence of malignancy in this pathology. Longitudinal studies of CD patients should be performed to confirm this evidence.

Nucleolar organizer function in developing potato calli

The number of transcriptionally active nucleolar organizer regions (NORs) of one monohaploid, one dihaploid and two tetraploids of Solanum tuberosum and one diploid S. phureja was established by the silver staining of metaphases in root meristems and in in vitro-cultured leaf explants. The maximum number of active NORs per cell was one per haploid set of chromosomes. One or more NORs could be inactive in cells of the tetraploid meristems and in non-polyploidized and polyploidized cells of the dihaploid and tetraploid explants. Inactivation was determined by genotype and tissue and could remain constant during in vitro culture.

A nucleolus organizer region in a B chromosome inactivated by DNA methylation

Chromosomal DNA methylation patterns were determined in the grasshopper Eyprepocnemis plorans by in situ digestion with MspI and HpaII. While no methylated regions were observed in standard chromosomes, the B chromosome was methylated in the distal third of its long arm. In this zone the B chromosome had an active nucleolus organizer region (NOR) in a male carrying a centric fusion between the B and the longest autosome, and it was not methylated. This NOR, however, was never observed in the active form in nonfused B chromosome, possibly because of methylation of this B chromosome region.

Characterization of precancerous and neoplastic human testicular germ cells.

Testicular germ cell tumors are in most cases preceded by an intratubular noninvasive form of neoplasia, known as carcinoma in situ (CIS, Skakkebaek and Berthelsen 1978). These atypical germ cells resemble gonocytes or spermatogonia (Nüesch-Bachmann and Hedinger 1977), but also show nuclear atypia indicative of malignant transformation. Atypical intratubular cells have been known to precede overt tumors for up to 16 years (Bannwart et al. 1988). However, they do not have invasive properties and the attempts to grow them as xenografts in nude mice have been unsuccessful (Walt and Stevens unpublished results). The transition of CIS to invasive carcinoma therefore remains poorly understood. In an effort to understand better the pathobiology of testicular neoplasias and their precancerous precursors, we have studied the expression of certain biochemical tumor markers (Walt et al. 1986) and of alkaline phosphatase isozymes (Hofmann et al. 1989), the activity of nucleolus organizer regions (NORs) (Delozier-Blanchet et al. 1986a), and the presence of a number of specific chromosomal regions by in situ hybridization on paraffin sections (Emmerich et al. 1989). For these investigations we have used xenografts and their related cell lines (DelozierBlanchet et al. 1986b; Emmerich et al. 1989; Hofmann et al. 1989; Walt et al. 1986) as well as primary tumors and primary precancerous and ejaculated germ cells (Jauch et al. 1989; Walt et al. 1989). We hope that this summary of data will allow the development of new strategies for further research in this area.KeywordsGerm CellGerm Cell TumorEmbryonal CarcinomaTesticular Germ Cell TumorTesticular Germ CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A combined AgNOR-Feulgen staining technique.

We describe a new staining technique (H-Ag-S) which allows observation and counting of active nucleolus organizer regions (NORs) and evaluation of the amount of DNA in the same cell nucleus. The procedure consists of combining a modified AgNOR staining method with the Feulgen reaction. A sequential procedure is proposed, based on the determination of optimal staining conditions. The technique, which was designed to allow studies of correlations between the transcriptional activity of rDNA genes and the cell ploidy, was primarily developed for rat liver smears. It should be applicable to most biological preparations, but the optimal conditions might be variable.

Culture of PNKT-4B cells at invasion permissive and restrictive temperatures. I. Chromosomal analysis

PNKT-4B is an aneuploid cell line derived from a herpesvirus-induced renal adenocarcinoma of Rana pipiens that displays restricted invasion at 21 degrees C or cooler and invasion at 23 degrees C through 28 degrees C. Metaphase chromosomes obtained from subcultures (passages 297; 345-347) grown at 18 degrees C or 28 degrees C were Giemsa stained or N-banded with acidic silver nitrate. Cells grown at 18 degrees C displayed a modal chromosome number of 41, while 28 degrees C cultures displayed a modal number of 40. The distribution of the chromosomes suggests that the two temperatures may be allowing growth of different subclonal populations. N-banding of chromosomes at both temperatures revealed an increase of active nucleolar organizer regions (NORs) over normal frog tissues, 2/2N. Analysis of 200 N-banded spreads from cells grown at each temperature revealed modal numbers of 9 NORs/cell and modal numbers of 6 NOR-containing chromosomes/cell. Nine specific NOR-containing chromosomes were identified and scored. Similar distributions were observed at 18 degrees C and 28 degrees C. The data imply that the modal number of PNKT-4B has shifted since it was first described, 39, and differs at invasion-permissive and -restrictive temperatures. Increased numbers of active NORs and alterations of NOR-containing chromosomes imply an amplification of rDNA over the amount in normal frog.