Three-dimensional organization of ribosomal DNA in interphase nuclei ofPisum sativum by in situ hybridization and optical tomography

The three-dimensional (3D) organization of rDNA-containing chromatin was studied in structurally well preserved, interphase nuclei ofPisum sativum root tips by in situ hybridization using a biotinylated cDNA probe to the 18, 5.8 and 25 S rDNA sequences. The probe was detected by immunofluorescence and optical section images recorded either by video imaging or by using a confocal laser scanning microscope. Detailed 3D reconstructions were made of 12 nucleoli by projection of confocal optical sections. The probe labelled four perinucleolar heterochromatin sites, one pair 1.0–2.1 µm in diameter and the other 0.5–1.0 µm diameter. It also labelled intranucleolar structures including 300–500 nm spots emanating from the perinucleolar sites into the body of the nucleolus. The intranucleolar labelled structures emanating from the perinucleolar sites lay in discrete domains. Medium power observations of 22 fields of cells (6–30 cells per field) were made by optical sectioning using a video camera and computer deblurring. The arrangement of the perinucleolar sites was modelled in each cell and the arrangements examined for nonrandomness. The sites tended to be spaced out around the nucleolar periphery approximating a regular tetrahedral arrangement as if to minimize clustering and the large sites appeared to lie in a plane perpendicular to the root axis. Cells with multiple nucleoli did not have any preferred distribution of sites between nucleoli. These observations are discussed in terms of current models of rDNA organization.     

Cytological localization of inverted repeated DNA sequences in Vicia faba

Inverted repeated DNA sequences have been isolated from sheared Vicia faba DNA by hydroxylapatite column chromatography, treated with nuclease S₁, tritiated by the nick translation method and hybridized in situ on squashes of Vicia faba root tips. Silver grains appear grouped in a rather limited portion of interphase nuclei and form a sort of band across them. The central regions of metaphase chromosomes are preferentially labeled, labeling being excluded from telomeres, centromeres and secondary constrictions. These results are briefly discussed in relation to those obtained in other species and the functional significance of inverted repeats.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Nuclear DNA characterization of two species ofVicia:Vicia bithynica L. andVicia narbonensis L.

The speciesVicia bithynica andVicia narbonensis, from the same subgeneric section ofVicia faba, show variations in nuclear DNA content Nuclear DNAs, extracted from root tips of the twoVicia species, were characterized by thermal denaturation, analytical ultracentrifugation and reassociation kinetics. The thermal denaturations of DNA, the number of DNA components reassociating with second order kinetics, the proportion of repeated DNA sequences, the frequency of the repeated DNA classes are reported and compared to previous data onVicia faba DNA. Feulgen absorptions at different thresholds of optical density⁺ of interphase nuclei in cytological preparations of the root meristems ofV. bithynica andV. narbonensis are determined and compared withV. faba analogous determinations. The results, confirming that plant genome is highly flexible, are discussed in relation to other data on the interspecific variations of the nuclear DNA content.     

Location of the genes for 5S ribosomal RNA in Xenopus laevis

In situ hybridization of 5S RNA and cRNA transcribed in vitro from Xenopus laevis 5S DNA shows that 5S DNA is localized at or near the telomere region of the long arm of many, if not all, of the X. laevis chromosomes. No 5S DNA is detected near the nucleolus organizer in the normal X. laevis chromosome complement, but in a X. laevis kidney cell line, 5S DNA is found at the distal end of the secondary constriction. The arrangement of 5S DNA in several types of interphase nuclei is described. — During the pairing stages of meiosis the telomeres of most or perhaps all of the chromosomes become closely associated so that the regions containing 5S DNA form a single cluster. This close association might be either a cause or a result of the presence of the similar sequences of 5S DNA on many telomeres. It suggests that the uniformity of 5S sequences on non-homologous chromosomes might be maintained by crossing-over between the chromosomes.     

Nuclear DNA variability withinPisum sativum (Leguminosae): Cytophotometric analyses

The cultivars or experimental lines ofPisum sativum were analyzed cytophotometrically for nuclear DNA content of early prophases after ethanol-acetic acid fixation. Wide variability was found (from 3.93 to 5.07pg per haploid nucleus). This result was confirmed by the cytophotometric analysis of interphase nuclei isolated from leaf tissues fixed in formalin. Analysis of interphase nuclei at different thresholds of optical density showed that certain chromatin fractions are involved in the variations.     

Chromatin arrangements in intact interphase nuclei examined by laser confocal microscopy

Using a laser confocal microscope, chromatin arrangements in intact interphase nuclei were investigated in four plant species. Chromosomes in these plants have specific segments that can be stained with the fluorescent dye chromomycin A₃ (CMA). We stained centromeres inHordeum vulgare, sub-telomeric regions inSecale cereale, satellites inChrysanthemum multicore, and the satellites and the short arms of chromosomes with satellites inHemerocallis middendorfii. The following points were shown: (1) In mitotic interphase nuclei, the centromere and the telomeres of both arms touched the nuclear membrane and had evident polarity. Some CMA-bodies in sub-telomeric regions do not contact the nuclear membrane. (2) Differentiated nuclei had a non-random construction. Polarity of chromosomes is maintained, however, the chromosomes are far apart from the nuclear membrane. (3) Associations in sub-telomeric regions in the interphase nuclei ofSecale cereale were probably due to the association of heterochromatic regions with identical repeated sequences rather than telomere associlations. (4) In interphase nuclei ofChrysanthemum multicore, satellites fused during interphase.     

Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

 The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS), a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS, C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection of low-copy DNA sequences. Received: 17 September 1996 / Accepted: 18 October 1996     

Orientation of interphase chromosomes as detected by Giemsa C-bands

The orientation of Giemsa C-bands has been studied in mitotic and interphase cells of Allium cepa, A. sativum and of Aloe vera. The C-bands in these three species are located at the telomeres, secondary constriction region of the nucleolar chromosomes and the centromeric regions, respectively. Observations in A. cepa and Aloe indicate clearly that the interphase chromosomes are non-random in their orientation and possibly maintain their telophase configuration through the attachment of telomeres and perhaps of kinetochores with the nuclear membrane. Electron micrographs of onion cells also reveal that certain heterochromatic segments are associated with the nuclear membrane. — The nucleolar interstitial C-bands in A. sativum remain free in the nucleoplasm and may come close to each other due to heterochromatic attraction. Such a heterochromatic attraction is also evident between telomeric regions and between centromeres. However, a two by two attachment could not be noticed. A diagrammatic representation of the orientation of interphase chromosomes has been presented.The major part of this work was presented at the First International Congress on Cell Biology, Boston, Sept. 5–10, 1976 (Platform Session 36, J. Cell Biol. 70, 418a (1976)     

The possible presence of aldehydes and carbohydrates in chromosomes and interphase nuclei

Summary A histochemical study of the mitotic chromosomes and interphase nuclei of rat regenerating liver and of root tips from Vicia faba and Trillium grandiflorum has been performed using the periodic acid-Schiff reaction. Carbohydrates and plasmalogen-like molecules were demonstrated on these cellular structures. The presence of such components was confirmed by biochemical studies upon nuclei isolated from rat liver.     

Effect of Telomeres on the Interphase Location of Adjacent Regions of the Human X Chromosome

Chromosomes occupy specific nonrandom domains in the interphase nucleus of eukaryotic cells. We have used a Chinese hamster-human somatic cell hybrid line containing a single human X chromosome to study the interphase distribution of the Xp telomere using fluorescent in situ hybridization and optical sectioning. A derivative cell line in which the X chromosome has been broken at Xq22-24 and healed by the addition of cloned human telomeric sequences was also studied to determine if introduction of these sequences at a previously interstitial site changed its location in interphase. The endogenous Xp telomere occupies a specific, nonrandom, internal domain. Introduction of a telomere at a previously interstitial site did not alter the interphase nuclear location of that site. The results suggest that nonrandom interphase location of telomeres may not be determined solely by the DNA sequence of the telomere.     

Localization of Tubulin and a Centrin-Homologue in Vegetative Cells and Developing Gametangia of Ectocarpus siliculosus (Dillw.) Lyngb. (Phaeophyceae,Ectocarpales)

The distribution of tubulin and centrin in vegetative cells and during gametogenesis of Ectocarpus siliculosus was studied by immunofluorescence. In interphase cells bundles of microtubules are focused on the centriolar region near the nuclear surface. Some of the bundles ensheath the nucleus while others traverse the cytoplasm in various directions, sometimes reaching the cell cortex. Evaluation of serial optical sections by confocal laser scanning microscopy (CLSM) revealed that the perinuclear and “cytoplasmic” microtubule bundles presumably constitute a single complex. In interphase cells centrin is localized as a single bright spot in the centriolar region. In dividing cells duplication and separation of the microtubular complex and the centrin spot takes place. In post-mitotic cells with two nuclei, the centrioles are located at opposite cell poles, short microtubule bundles emanate from them and partially encompass the nucleus. During gametogenesis a gradual transformation of the vegetative cytoskeleton to the gametic flagellar apparatus occurs.     

3D nuclear organization of telomeres in the Hodgkin cell lines U-HO1 and U-HO1-PTPN1: PTPN1 expression prevents the formation of very short telomeres including "t-stumps"

Background  

In cancer cells the three-dimensional (3D) telomere organization of interphase nuclei into a telomeric disk is heavily distorted and aggregates are found. In Hodgkin's lymphoma quantitative FISH (3D Q-FISH) reveals a major impact of nuclear telomere dynamics during the transition form mononuclear Hodgkin (H) to diagnostic multinuclear Reed-Sternberg (RS) cells. In vitro and in vivo formation of RS-cells is associated with the increase of very short telomeres including "t-stumps", telomere loss, telomeric aggregate formation and the generation of "ghost nuclei".     

Reversible chromosome condensation induced in Drosophila embryos by anoxia: visualization of interphase nuclear organization

We have studied the morphology of nuclei in Drosophila embryos during the syncytial blastoderm stages. Nuclei in living embryos were viewed with differential interference-contrast optics; in addition, both isolated nuclei and fixed preparations of whole embryos were examined after staining with a DNA-specific fluorescent dye. We find that: (a) The nuclear volumes increase dramatically during interphase and then decrease during prophase of each nuclear cycle, with the magnitude of the nuclear volume increase being greatest for those cycles with the shortest interphase. (b) Oxygen deprivation of embryos produces a rapid developmental arrest that is reversible upon reaeration. During this arrest, interphase chromosomes condense against the nuclear envelope and the nuclear volumes increase dramatically. In these nuclei, individual chromosomes are clearly visible, and each condensed chromosome can be seen to adhere along its entire length to the inner surface of the swollen nuclear envelope, leaving the lumen of the nucleus devoid of DNA. (c) In each interphase nucleus the chromosomes are oriented in the "telophase configuration," with all centromeres and all telomeres at opposite poles of the nucleus; all nuclei at the embryo periphery (with the exception of the pole cell nuclei) are oriented with their centromeric poles pointing to the embryo exterior.     

A proposed model of chromosomal organization in nuclei at fertilization

Evidence is presented to support the proposition that the position of chromosomes within nuclei is determined by the following factors: (1) the location of centromeres on one side of the nucleus and telomeres(ends) on the other (reflecting the telophase orientation brought about by their poleward anaphase migration); (2) attachment of telomeres to the nuclear membrane (site of attachment in relation to the poles and equator being dependent on the length of the individual arms and point 1 above); (3) telomere-to-telomere attachment of nonhomologues in a specific sequence; (4) telomere-to-telomere attachment of certain homologous chromosomes.It is proposed that a specific arrangement of nonhomologues occurs within gametic nuclei following meiosis, while initial homologous alignment takes place during karyogamy (fusion of gametic nuclei). The method of homologous association of telomeres is dependent on whether or not karyogamy within the species is between interphase pronuclei or occurs during the first cleavage division. A model of chromosome behavior for both these type of karyogamy is presented.     

Constitutive heterochromatin distribution in monocentric and polycentric chromosomes

Monocentric chromosomes of Vicia faba (2n=12) and polycentric chromosomes of Luzula purpurea (2n=6) and L. multiflora (2n=36) were studied by the C-banding technique. C-positive regions exhibited a restricted distribution in V. faba nuclei and were located near the centromeres of the chromosomes. Each chromosome had both a characteristic number and distribution of C-positive regions permitting homologue identification. L. purpurea and L. multiflora C-bands were much more numerous than those of V. faba and were widely distributed throughout both nuclei and chromosomes. Three distinct constitutive heterochromatin distribution patterns were present in L. purpurea metaphase chromosomes permitting homologous chromosome identification. One of three C-band distribution patterns was also evident in L. multiflora chromosomes.     

Three-dimensional organization of interphase fibroblast nuclei in two closely related shrew species (<Emphasis Type="Italic">Sorex granarius</Emphasis> and <Emphasis Type="Italic">Sorex araneus</Emphasis>) differing in the structures of their chromosome termini

A FISH with a probe for telomeric and rDNA repeats and immunofluorescence with ANA CREST and antibodies to nucleolae protein B23 were used to study the three-dimensional (3D) organization of fibroblast interphase nuclei in two shrew twin species, Sorex granarius and Sorex araneus, of the Cordon race. Karyotypes of these species are composed of nearly identical chromosomal arms and differ in the number of their metacentrics and the structures of their terminal chromosome regions. In the short arms of S. granarius, 32 of the acrocentrics have large telomeres that contain an average of 218 kb telomere repeats, which alternate with ribosomal repeats. These regions also contain active nucleolar organizing regions (NORs). In contrast, in active NORs in S. araneus are localized at the terminal regions of 8 chromosomal arms (Zhdanova et al., 2005; 2007b). Here, we show that associations of chromosomes by telomeres and the contact of a part of the telomere clusters with the inner nuclear membrane and nucleolus characterize the interphase nuclei of both Sorex granarius and Sorex araneus. We also reveal the partial colocalization of telomere and ribosomal clusters and the spatial proximity of centomeric and telomeric regions in the interphase nuclei of S. granarius. It appears that only ribosomal clusters containing a sufficient number of active ribosomal genes exhibit a connection with the nucleolus. Nucleolus disassembly during the fibroblastís transition to mitosis and the role of the B23 protein in this process have been studied.     

The role of microtubules in the maintenance of regular localization and arrangement of Golgi apparatus in root cells of Triticum aestivum L.

In plant cells Golgi apparatus organization, maintenance and distribution differ from that in mammalian cells and the mechanisms for this are not clearly understood. Here we investigate the role of microtubules in the positioning and arrangement of Golgi apparatus in the root cells of Triticum aestivum L. by using dual immunofluorescent labeling and laser confocal microscopy to localize both throughout the cell cycle. We observed that Golgi stacks (i) in interphase cells predominantly occupied the perinuclear region, (ii) during mitosis they redistributed to the spindle periphery and/or areas above spindle poles, and (iii) in telophase accumulated around the phragmoplast and the chromosomes/nuclei of daughter cells. Inhibition of microtubule assembly by colchicine resulted in aggregation of Golgi in the cortical cytoplasm of interphase cells and accumulation around the chromosomes in C-mitotic cells, in stark contrast with the distribution in untreated cells. Electron microscopy revealed that in colchicine treated cells many Golgi units became disorganized, yet others were abnormally enlarged. Overall, our results indicate that in plant cells microtubules play a key role in restricting the position and maintaining the arrangement and structural integrity of the Golgi apparatus.     

Determination of 4-chloroindole-3-acetic acid methyl ester in Lathyrus, Vicia and Pisum by gas chromatography – mass spectrometry

4-Chloroindole-3-acetic acid methyl ester was identified unequivocally in Lathyrus latifolius L., Vicia faba L. and Pisum sativum L. by thin layer chromatography, gas chromatography and mass spectrometry. The gas chromatographic system was able to separate underivatized chloroindole-3-acetic acid methyl ester isomers. The quantitative determination of 4-chloroindole-3-acetic acid methyl ester in immature seeds of these three species was performed by gas chromatography – mass spectrometry using deuterium labelled 4-chloro-indole-3-acetic acid methyl ester as an internal standard. P. sativum contained approximately 25 mg kg^-1, V. faba 1–2 mg kg^-1 and L. latifolius 2 mg kg^-1 dry weight.     

A family of dispersed repeats in the genome of Vicia faba: structure, chromosomal organization, redundancy modulation, and evolution

A family of repeated DNA sequences of about 1200 bp in length and bordered by well-conserved, 18 bp inverted repeats (VfB family) was found in the nuclear genome of Vicia faba. The structure, chromosomal organization, redundancy modulation and evolution of these sequences were investigated. They are enriched in A+T base pairs (about 40% G+C) and lack any obvious internally repeated motif. A 64%–73% nucleotide sequence identity was found when pairwise comparisons between VfB sequences were carried out (average 69%). Direct repeats were not found to flank the inverted repeats that border these DNA sequences. The results obtained by hybridizing VfB repeats to Southern blots of V. faba genomic DNA digested with EcoRI indicated that these DNA elements are interspersed in the genome. The appearance of bands in these Southern blots and comparison of the structure of the sequences that flank different VfB elements showed that these repeats might be part of other, longer repeated DNA sequences. A high degree of dispersion throughout the genome was confirmed by cytological hybridization, which showed VfB sequences to be scattered along the length of all chromosomes and to be absent or rare only at heterochromatic chromosomal regions. These sequences contribute to intraspecific alterations of genomic size. Indeed, dot-blot hybridizations proved that their redundancy, which is positively correlated with the overall amount of nuclear DNA in each accession, varies between V. faba land races (27×10³–230×10³ copies per 1C DNA). Southern blot hybridization of VfB repeats to restriction endonuclease-digested genomic DNAs of V. faba, V. narbonensis, V. sativa, Phaseolus coccineus, Populus deltoides, and Triticum durum revealed nucleotide sequence homology of these DNA elements, whatever the stringency conditions, only to the DNAs of Vicia species, and to a reduced extent to the DNAs of V. narbonensis and V. sativa compared with that of V. faba. It is concluded that VfB repeats might be descended from mobile DNA elements and contribute to change genomic size and organization during evolution. Received: 10 September 1998; in revised form: 12 May 1999 / Accepted: 19 May 1999