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

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Abstract

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.

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References

  • Agard DA (1984) Optical sectioning microscopy: cellular architecture in three dimensions. Annu Rev Biophys Bioeng 13:191–219

    Google Scholar 

  • Agard DA, Sedat JW (1983) Three dimensional architecture of a polytene nucleus. Nature 302:676–681

    Google Scholar 

  • Agard DA, Hiraoka Y, Shaw PJ, Sedat JW (1989) Fluorescence microscopy in three dimensions. Methods Cell Biol 30:353–377

    Google Scholar 

  • Arroua M-L, Hartung M, Devictor M, Berge-Lefranc J-L, Stahl A (1982) Localisation of ribosomal genes by in situ hybridisation in the fibrillar centre of the nucleolus in the human spermatocyte. Biol Cell 44:337–340

    Google Scholar 

  • Avivi L, Feldman M (1980) Arrangement of chromosomes in the interphase nucleus of plants. Hum Genet 55:281–295

    Google Scholar 

  • Castleman KR (1979) Optical sectioning. In: Digital image processing. Prentice-Hall, New Jersey, USA, pp 351–360

    Google Scholar 

  • Cataldo C, Souchier C, Stahl A (1988) Three-dimensional ultrastructure and quantitative analysis of the human Sertoli cell nucleolus. Biol Cell 63:277–285

    Google Scholar 

  • Ellis THN, Davies DR, Castleton JA, Bedford ID (1984) The organization and genetics of rDNA length variants in peas. Chromosoma 91:74–81

    Google Scholar 

  • Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13

    Google Scholar 

  • Feinberg AP, Vogelstein B (1984) Addendum: A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266–267

    Google Scholar 

  • Flavell RB (1986) The structure and control of expression of ribosomal RNA genes. In: Miflin BJ (ed) Plant molecular and cell biology, vol 3. Oxford University Press, Oxford, pp 251–274

    Google Scholar 

  • Ghosh S, Paweletz N (1987) Active ribosomal cistrons and their primary transcripts in the nucleolus. Cell Biol Int Rep 11:205–210

    Google Scholar 

  • Goessens G (1984) Nucleolar structure. Int Rev Cytol 87:107–158

    Google Scholar 

  • Hochstrasser M, Mathog D, Gruenbaum Y, Saumweber H, Sedat JW (1986) Spatial organization of chromosomes in the salivary gland nuclei ofDrosophila melanogaster. J Cell Biol 102:112–123

    Google Scholar 

  • Jordan EG (1987a) Nucleolar organisers in plants. Chromosomes Today 9:272–283

    Google Scholar 

  • Jordan EG (1987b) At the heart of the nucleolus. Nature 329:489–490

    Google Scholar 

  • Jordan EG, Chapman JM (1971) Ultrastructural changes in the nucleoli of Jerusalem artichoke (Helianthus tuberosus) tuber discs. J Exp Bot 22:627–634

    Google Scholar 

  • Jordan EG, Chapman JM (1973) Nucleolar and nuclear envelope ultrastructure in relation to cell activity in discs of carrot root (Daucus carota). J Exp Bot 24:197–209

    Google Scholar 

  • Jordan EG, Bennett MD, Smith JB (1982a) Nucleolar organizers and fibrillar centres inTriticum aestivum L., cv chinese spring. Chromosoma 87:447–559

    Google Scholar 

  • Jordan EG, Martini G, Bennett MD, Flavell RB (1982b) Nucleolar fusion in wheat. J Cell Sci 56:485–495

    Google Scholar 

  • Jorgensen RA, Cuellar RE, Thompson WF, Kavanogh TA (1987) Structure and variation in ribosomal RNA genes of Pea. Plant Mol Biol 8:3–12

    Google Scholar 

  • Lloyd CW, Wells B (1985) Microtubules are at the tip of root hairs and form helical patterns corresponding to inner wall fibrils. J Cell Sci 75:225–238

    Google Scholar 

  • Manuelidis L (1985a) Indications of centromere movement during interphase and differentiation. Ann NY Acad Sci 450:205–221

    Google Scholar 

  • Manuelidis L (1985b) Individual interphase chromosome domains revealed by in situ hybridisation. Hum Genet 71:288–293

    Google Scholar 

  • Manuelidis L, Borden L (1988) Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction. Chromosoma 96:397–410

    Google Scholar 

  • Mathog D (1985) Light microscope based analysis of three-dimensional structure: applications to the study of Drosophila salivary gland nuclei. II. Algorithms for model analysis. J Microsc 137:253–273

    Google Scholar 

  • Mathog D, Hochstrasser M, Sedat JW (1985) Light microscope based analysis of three-dimensional structure: applications to the study of Drosophila salivary gland nuclei. I. Data collection and analysis. J Microsc 137:241–252

    Google Scholar 

  • Motte P, Deltour R, Mosen H, Bronchart R (1988) Three-dimensional electron microscopy of the nucleolus and nucleolus-associated chromatin (NAC) during early germination of Zea mays L. Biol Cell 62:65–81

    Google Scholar 

  • Raska I, Reimer G, Jarnik M, Kostruch Z, Raska K (1989) Does the synthesis of ribosomal RNA take place within nucleolar fibrillar centers or dense fibrillar components? Biol Cell 65:79–82

    Google Scholar 

  • Rawlins DJ, Shaw PJ (1988) Three-dimensional organization of chromosomes ofCrepis capillaris by optical tomography. J Cell Sci 91:401–414

    Google Scholar 

  • Richmond TJ, Finch JT, Rushton B, Rhodes D, Klug A (1984) Structure of the nucleosome core particle at 7A resolution. Nature 311:532–537

    Google Scholar 

  • Scheer U, Rose KM (1984) Localization of RNA polymerase I in interphase cells and mitotic chromosomes by light and electron microscopic immunocytochemistry. Proc Natl Acad Sci USA 81:1431–1435

    Google Scholar 

  • Simpson PR, Newman M, Davies DR (1988) Detection of legumin gene DNA sequences in pea by in situ hybridization. Chromosoma 96:454–458

    Google Scholar 

  • Thiry M (1988) Immunoelectron microscope localization of bromodeoxyuridine incorporated into DNA of Ehrlich tumor cell nuclei. Exp Cell Res 179:204–213

    Google Scholar 

  • Thiry M, Scheer U, Goessens G (1988) Immunoelectron microscopic study of nucleolar DNA during mitosis in Ehrlich tumor cells. Eur J Cell Biol 47:346–357

    Google Scholar 

  • Thiry M, Thiry-Blaise L (1989) In situ hybridization at the electron microscope level: an improved method for precise localization of ribosomal DNA and RNA. Eur J Cell Biol 50:235–243

    Google Scholar 

  • Troster H, Spring H, Meissner B, Schultz P, Oudet P, Trendelenburg MF (1985) Structural organization of an active, chromosomal nucleolar organizer region (NOR) identified by light microscopy and subsequent TEM and STEM electron microscopy. Chromosoma 91:151–163

    Google Scholar 

  • van Dekken H, Pinkel D, Mullikin J, Trask B, van den Engh G, Gray J (1989) Three-dimensional analysis of the organization of human chromosome domains in human and humanhamster hybrid interphase nuclei. J Cell Sci 94:299–306

    Google Scholar 

  • Wachtler F, Hartung M, Devictor M, Wiegant J, Stahl A, Schwarzacher HG (1989) Ribosomal DNA is located and transcribed in the dense fibrillar component of human Sertoli cell nucleoli. Exp Cell Res 184:61–71

    Google Scholar 

  • Weinstein M, Castleman KR (1971) Reconstructing 3-d specimens from 2-D section images. Proc Soc Photo-opt Instr Eng 26:131–138

    Google Scholar 

  • White JG, Amos WB, Fordham M (1987) An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy. J Cell Biol 105:41–48

    Google Scholar 

  • Wolff G, Quednau HD (1988) The arrangement of Pisum chromosomes in interphase. Genome 30:717–722

    Google Scholar 

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  1. John Innes Institute and AFRC Institute of Plant Science Research, Colney Lane, NR4 7UH, Norwich, UK

    David J. Rawlins & Peter J. Shaw

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  1. David J. Rawlins
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  2. Peter J. Shaw
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Rawlins, D.J., Shaw, P.J. Three-dimensional organization of ribosomal DNA in interphase nuclei ofPisum sativum by in situ hybridization and optical tomography. Chromosoma 99, 143–151 (1990). https://doi.org/10.1007/BF01735331

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  • DOI: https://doi.org/10.1007/BF01735331

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