Chromosomal constitution of nucleolus-associated chromatin in man

Summary Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.     

‘Nucleolar dominance’ as observed in barley translocation lines with specifically reconstructed SAT chromosomes

Summary Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.     

Mapping of pachytene bivalents of female codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae)

Spread pachytene nuclei of codling moth Cydia pomonella (Linnaeus) (Lep., Tortricidae) females of a Syrian strain (SY) were used to investigate chromomere patterns of chromosome bivalents and determine their length. The karyotype of female codling moths consists of 28 chromosome bivalents, of which seven are clearly distinguishable using chromosome length and the number and size of the chromomeres in the pachytene stage. One autosome bivalent has two nucleolar organizing regions (NORs) that are located at the opposite ends of the chromosome and appear as distinct structural landmarks. In female codling moths, the WZ sex chromosome bivalent was easily identified in pachytene oocytes according to the heterochromatic thread of the W chromosome. This study contributed to the knowledge and identification of pachytene chromosomes of female codling moths.     

Pachytene variations in Ricinus

The pachytene complement in microsporocytes was compared among ten races of Ricinus communis L., the castor plant (2n=20). Four kinds of pachytene variations were observed: (1) variation in degree of spreading of the chromosomes; (2) variation in degree of attenuation of heterochromatin; (3) variation in morphology, which appeared to be restricted to the two nucleolar organizing bivalents; (4) variation in frequency of association of each of these two bivalents with the nucleolus. It is suggested that variations in the pachytene chromosomes occur ubiquitously in this species.     

Nucleolar organizing region in theApiaceae (Umbelliferae)

Data available for the nucleolus organizing region in the familyApiaceae are reviewed. An attempt has been made to establish the exact number of this region in various subfamilies and tribes through studies on the karyotype and nucleolus. Most of the taxa have a single nucleolar chromosome per haploid complement. The location of the nucleolar organizing region (NOR) on the chromosome varies. Members ofHydrocotyloideae differ drastically from those of subfam.Saniculoideae andApioideae, with respect to the location of NOR. Despite wide geographical distribution, varied ecological preferences and differences in morphology, anatomy and cytology, Umbellifers have attained stability in the number and location of NORs. Characters of NOR offer scope for utilization in understanding phylogenetic relationships at higher levels of taxonomic hierarchy.Studies on the Nucleolus and Nucleolar Chromosomes in Angiosperms XII.     

Nucleolar behavior during meiosis in four species of phyllostomid bats (Chiroptera, Mammalia)

We analyzed the behavior of the nucleolus, nucleolar structures and nucleolus organizer regions (NORs) during meiotic division in four species of phyllostomid bats that have different numbers and locations of NORs. Nucleoli began disassembly at leptotene, and the subcomponents released from the nucleolus were dispersed in the nucleoplasm, associated with perichromosomal regions, or they remained associated with NORs throughout division. In Phyllostomus discolor, a delay in nucleolus disassembly was observed; it disassembled by the end of pachytene. The RNA complexes identified by acridine orange staining were observed dispersed in the nucleoplasm and associated with perichromosomal regions. FISH with rDNA probe revealed the number of NORs of the species: one NOR in Carollia perspicillata, one pair in Platyrrhinus lineatus and P. discolor, and three pairs in Artibeus lituratus. During pachytene, there was a temporary dissociation of the homologous NORs, which returned to pairing at diplotene. The variation in the number (from one to three pairs) and location of NORs (in sex or autosomal chromosomes, at terminal or interstitial regions) did not seem to interfere with the nucleolar behavior of the different species because no variation in nucleolar behavior that could be correlated with the variation in the number and chromosomal location of NORs was detected.     

Chromosome banding in Amphibia

The distribution and quantity of constitutive heterochromatin and of the nucleolus organizer regions (NORs) on the chromosomes of 22 species of bufonids and hylids (Amphibia, Anura) was investigated. Three different kinds of constitutive heterochromatin were found and the frequency of brightly fluorescing heterochromatic regions was remarkably high. On almost all chromosomes there is centric and telomeric heterochromatin. Quantitative estimates of heterochromatin demonstrate that large DNA differences among closely related species can not be attributed to differing quantities of constitutive heterochromatin. In all species investigated, only one homologous pair of NORs was found, which lies preferentially in the proximal and interstitial segments of the long chromosome arms. The NORs are always associated with constitutive heterochromatin on both sides. The size variability between homologous NORs is very high. In the euchromatic regions of the metaphase chromosomes, neither Q- nor G-bands can be demonstrated; this can be attributed to an extremely strong contraction of the anuran chromosomes. On the basis of these results various mechanism of the chromosomal evolution in Anura are discussed.     

The behavior of nucleolus organizers in structurally changed karyotypes of barley

Two standard karyotype barley lines and 18 lines with karyotypes reconstructed by means of induced reciprocal translocations have been studied with respect to nucleolus formation. The standard karyotype contains two pairs of satellite chromosomes (pairs 6 and 7). Five of the structurally changed karyotypes contain, as a result of reciprocal translocations between the standard satellite chromosomes, only one satellite chromosome pair, each chromosome with two satellites and two nucleolus organizing regions. Under these circumstances, only two of the four NORs are active in nucleolus formation while the other two — probably the transposed ones — remain inactive; hence the maximum number of primary nucleoli per nucleus is two. — When NORs are translocated to chromosomes with no NOR in the standard karyotyp, the normal pattern of nucleolus formation remains unchanged. The same is true after transposition of segments from other chromosomes to the satellites of the standard SAT-chromosome pairs 6 and 7. The results obtained are discussed with respect to effects of translocations on the activity and behaviour of nucleolus organizing regions.     

NOR Sites Detected by Ag-DAPI Staining of an Unusual Autosome Chromosome of Bradysia Hygida (Diptera:Sciaridae) Colocalize with C-banded Heterochromatic Region

The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

A highly reproducible method of nucleolus organizing regions staining in plants

A method for the specific detection of the nucleolus organizing regions (NORs) of plant chromosomes has been developed employing enzymatic maceration and successive flame-drying for chromosome spreading and incubation with aqueous 50% AgNO3 at 55-60 C. When this method was applied to metaphase chromosomes the NORs were specifically discriminated as heavily stained segments in all the plant species examined. In the satisfactory results obtained by monitoring the reaction under a microscope during the course of the silver treatment, the chromosome arms were stained yellow to light brown while the NORs were dark brown to black. The present method has the advantage of yielding highly reproducible results for the specific detection of the NORs in plant materials.     

Position-dependent NOR activity in barley

Two types of intraspecific nucleolar dominance/suppression are described for barley,Hordeum vulgare L. When the nucleolus organizing regions (NORs) originally belonging to chromosomes 6 and 7 are combined by translocation in one chromosome, NOR 6 is dominant over NOR 7. Neither significant loss of rDNA nor its hypermethylation is the reason for the reduced nucleolus forming activity of NOR 7. Intrachromosomal NOR suppression probably does not occur in isochromosome 6s, which has two NORs 6 in one chromosome. Meiotic and somatic pairing of the homologous arms might be the reason for early fusion of their nucleoli and thus for the lower than expected maximum number of interphase nucleoli. Variable suppression of a partial NOR (6³) is described for descendants of crosses between translocation lines with split NORs 6 and 7. In these cases also, the reduced activity of the partial NOR 6³ is not due to deletion of rDNA as shown by in situ hybridization. Unstable methylation of NOR 6³ in heterozygous F₁ individuals is probably the cause of this phenomenon.     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

Cytological localization of the genes for the four classes of ribosomal RNA (25S, 18S, 5.8S and 5S) in polytene chromosomes of Phaseolus coccineus

Homologous tritiated 25S, 18S and 5.8S rRNAs were used separately for in situ hybridization to the polytene chromosomes of the embryo suspensor cells of Phaseolus coccineus. Hybridization occurred at the same chromosomal sites which were labeled in previous in situ hybridization experiments with 25+18S rRNAs in the same material (Avanzi et al., 1972), namely: nucleolus organizing system (satellite, nucleolar constriction and organizer) of chromosome pairs I (S₁) and V (S₂), proximal heterochromatic segment of the long arm of chromosome pair I, and terminal heterochromatic segment of chromosome pair II. Competition hybridization experiments confirmed for P. coccineus the high sequence homology between 25S and 18S rRNA already known for other plants.Homologous ¹²⁵I-5S rRNA was found to hybridize to three sites in the polytene chromosomes of P. cocdneus: the proximal heterochromatic segment in the long arm of chromosome pair I (which also bears the sequences complementary to 25S, 18S and 5.8S RNAs), most of the proximal heterochromatic segment plus a small portion of adjoining euchromatin in the long arm of chromosome pair VI and the large intercalary heterochromatic segment in the same chromosome pair. Simultaneous labeling of the two 5S RNA sites in chromosome VI was quite rare (3%), the rule being labelling of one site to the exclusion of the other, with a labeling frequency of 43.7% and 53.3% for sites no. 1 and no. 2 respectively. These results are interpreted as being due to differential hybridizability of chromosomal sites such as described in other materials.     

Nucleolar assembly of the rRNA processing machinery in living cells

To understand how nuclear machineries are targeted to accurate locations during nuclear assembly, we investigated the pathway of the ribosomal RNA (rRNA) processing machinery towards ribosomal genes (nucleolar organizer regions [NORs]) at exit of mitosis. To follow in living cells two permanently transfected green fluorescence protein-tagged nucleolar proteins, fibrillarin and Nop52, from metaphase to G1, 4-D time-lapse microscopy was used. In early telophase, fibrillarin is concentrated simultaneously in prenucleolar bodies (PNBs) and NORs, whereas PNB-containing Nop52 forms later. These distinct PNBs assemble at the chromosome surface. Analysis of PNB movement does not reveal the migration of PNBs towards the nucleolus, but rather a directional flow between PNBs and between PNBs and the nucleolus, ensuring progressive delivery of proteins into nucleoli. This delivery appeared organized in morphologically distinct structures visible by electron microscopy, suggesting transfer of large complexes. We propose that the temporal order of PNB assembly and disassembly controls nucleolar delivery of these proteins, and that accumulation of processing complexes in the nucleolus is driven by pre-rRNA concentration. Initial nucleolar formation around competent NORs appears to be followed by regroupment of the NORs into a single nucleolus 1 h later to complete the nucleolar assembly. This demonstrates the formation of one functional domain by cooperative interactions between different chromosome territories.     

Nucleolus,nucleolar chromosomes,and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte

Summary The shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of³H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of³H-uridine uptake showed that the oocyte was not at a resting stage. The possible cytogenetic consequences of these observations are discussed.     

Color differential staining of NOR-associated heterochromatic segments using acridine orange

A new staining technique has been evaluated for detecting heterochromatic segments accompanying nucleolus organizing regions (NORs). This technique essentially consists of C-banding followed by acridine orange staining. When the technique was applied to five species of plants, the NOR-associated heterochromatic segments (NOR H-segments) were differentiated from other segments of the chromosomes as regions emitting yellowish green fluorescence. An incubation of at least 30 min in hot 2 x SSC was required to make the NOR H-segments emit yellowish green fluorescence in Nothoscordum fragrans. Fluorescence on other heterochromatic segments varied from reddish orange to bright yellow; euchromatic segments emitted orange or yellowish orange fluorescence. The technique permits identification of NOR H-segments throughout mitosis based on the characteristic fluorescent color.