The Nucleus — Cytological Methods and Isolation for Biochemical Studies

  • J. F. Jackson
Part of the Modern Methods of Plant Analysis book series (MOLMETHPLANT, volume 1)


Interest in the plant cell nucleus has heightened in recent times due to the rapid advances being made in the biochemistry of its DNA, RNA, and protein components. While the cytological observations on chromosome structure and behavior have held interest in the nucleus for some time, application of recombinant DNA technology, sequencing and hybridization techniques to chromosomal DNA have focussed attention on the nucleus even more. Studies on gene expression have also seen many advances recently and much has been learnt about the events of eukaryotic mRNA biogenesis (Nevins 1983). Recognition of promoters and sites of transcriptional initiation, poly(A) addition, RNA splicing, methylation and the process of nuclear-cytoplasmic transport have been investigated and all need nuclear preparations at some stage for further investigation in the eukaryotic organism. Possession of a well-defined nucleus is one of the criteria separating eukaryotes from prokaryotes, and it is the pathway of mRNA synthesis involving this RNA processing and transport out of the nucleus which sets the eukaryotes apart from the prokaryotes as much as the other cytologically recognized nuclear differences. There is then great interest in nuclear preparation for these and other biochemical studies. This chapter will deal in the main with the larger-scale preparation of nuclei from plant cells for these biochemical studies. The properties of the nucleus will be dealt with only in so far as it explains the various techniques exploiting those properties which are used in nuclear preparation. Particular attention will be given to different procedures adopted for nuclear preparations for different purposes. The development of efficient protoplast preparation methods from plant cells has had a great effect on organelle investigations in plants (see Wagner, this vol.) including nuclear studies.


Methyl Green Euglena Gracilis Plant Nucleus Cytological Method Feulgen Staining 
These 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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ansa OA, Bowyer JW, Shepherd RJ (1982) Evidence for replication of cauliflower mosaicvirus DNA in plant nuclei. Virology 121:147–156PubMedCrossRefGoogle Scholar
  2. Arfman HA, Willmitzer L (1982) Endogenous protein kinase activity of tobacco nuclei: comparison of transformed, nontransformed cell cultures and the intact plant of Nicotiana Plant Sci Lett 26:31–38CrossRefGoogle Scholar
  3. Armstrong JA (1956) Histochemical differentiation of nucleic acids by means of induced fluorescence. Exp Cell Res 11:640–643PubMedCrossRefGoogle Scholar
  4. Atkinson MR, Jackson JF, Morton RK (1961) Nicotinamide mononucleotide adenylyl transferase of pig liver nuclei. Biochem J 80:318–323PubMedGoogle Scholar
  5. Belling J (1921) On counting chromosomes in pollen-mother cells. Am Nat 55:573–574CrossRefGoogle Scholar
  6. Bennet MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Philos Trans R Soc Lond B 274:227–274CrossRefGoogle Scholar
  7. Berlyn GP, Cecich RA (1976) Optical techniques for measuring DNA quantity. In: Mi-kache JP (ed) Modern methods in forest genetics. Springer, Berlin Heidelberg New YorkGoogle Scholar
  8. Bonner J (1976) The Nucleus. In: Bonner J, Varner JE (eds) Plant biochemistry, 3rd edn. Academic Press, New York, pp 37–64Google Scholar
  9. Bonner J, Chalkley GR, Dahmus M, Farnbrough D, Fujimura F, Huang RC, Huberman J, Jensen R, Marushige K, Ohlenbusch H, Olivera B, Widholm J (1968) Isolation and characterization of chromosomal nucleoproteins. Methods Enzymol 12:3–65CrossRefGoogle Scholar
  10. Bouman H, van Paridon H, Vogelaar A, Mennes AM, Libbenga KR (1981) Size analyses of RNA synthesized in isolated tobacco. Nicotiana tabacum cultivar white-burley callus nuclei. Plant Sci Lett 22:361–368CrossRefGoogle Scholar
  11. Branster MJ, Morton RK (1956) Comparative rates of synthesis of diphosphopyridine nucleotide by normal and tumour tissue from mouse mammary gland: studies with isolated nuclei. Biochem J 63:640–646PubMedGoogle Scholar
  12. Brewer EN (1979) Repair of radiation-induced DNA double-strand breaks in isolated nuclei of Physarum polycephalum Radiat Res 79:368–376PubMedCrossRefGoogle Scholar
  13. Brightwell MD, Leech CE, O’Farrell MK, Whish WJ, Shall S (1975) Poly ADP ribose polymerase in Polysarum polycephalum Biochem J 147:119–130PubMedGoogle Scholar
  14. Buetow DE (1976) Isolation of nuclei from protozoa and algae. In: Prescott DM (ed) Methods in cell biology. Academic Press, New York, pp 283–311Google Scholar
  15. Busch H, Smetana K (1970) The Nucleolus. Academic Press, New York, pp 548–575Google Scholar
  16. Caboche M, Lark KG (1981) Preferential replication of repeated DNA sequences in nuclei isolated from soybean cells grown in suspension culture. Proc Nat Acad Sci USA 78:1731–1735PubMedCrossRefGoogle Scholar
  17. Capesius I, Meyer Y (1977) Isolation of nuclei from protoplasts of orchids. Cytobiologie 15:485–490Google Scholar
  18. Chabot JF, Leopold AC (1982) Ultrastructural changes of membranes with hydration in soybean seeds. Am J Bot 69:623–633CrossRefGoogle Scholar
  19. Chauveau J, Moulé Y, Rouiller CH (1956) Isolation of pure and unaltered liver nuclei. Morphology and biochemical composition. Exp Cell Res 11:317–321Google Scholar
  20. Chen Y-M, Huang D-H, Lin S-F, Lin C-Y, Key JL (1983) Fractionation of nucleoli from auxin-treated soybean hypocotyl into nucleolar chromatin and preribosomal particles. Plant Physiol 73:746–753PubMedCrossRefGoogle Scholar
  21. Coleman AW (1978) Visualization of chloroplast DNA with two fluorochromes. Exp Cell Res 114:95–100PubMedCrossRefGoogle Scholar
  22. Dawson JRY, Thomas K, CI egg MT (1982) Purification of total cellular DNA from a single plant. Biochem Genet 20:209–219CrossRefGoogle Scholar
  23. Dhar AC, Shah CK (1982) Cytochemical method to localize acid nuclear proteins. Stain Technol 57:151–155PubMedGoogle Scholar
  24. Dhillon SS, Berlyn GP, Miksche JP (1977) Requirement of an internal standard for micro-spectrophotometric measurement of DNA. Am J Bot 64:117–121CrossRefGoogle Scholar
  25. Dhillon SS, Berlyn GP, Miksche JP (1978) Nuclear DNA content in populations of Pinusrigida Am J Bot 65:192–196CrossRefGoogle Scholar
  26. Dounce AL (1943) Enzyme studies on isolated cell nuclei of rat liver. J Biol Chem 147:685–698Google Scholar
  27. Dounce AL, Tishkoff GH, Barnett SR, Freer RM (1950) Free amino acids and nucleic acid content of cell nuclei isolated by a modification of Behrens technique. J Gen Physiol 33:629–642PubMedCrossRefGoogle Scholar
  28. Erdmann H, Boecher M, Wagner KG (1982) Two protein kinases from nuclei of cultured tobacco Nicotiana tabacum cultivar white-burley cells with properties similar to the cyclic nucleotide independent enzymes N-I and N-II from animal tissue. FEBS Lett 137:245–248PubMedCrossRefGoogle Scholar
  29. Franke WW (1974) Structure, biochemistry, and functions of the nuclear envelope. Int Rev Cytol [Suppl] 4:72–236Google Scholar
  30. Franklin AL, Filion WG (1981) Acridine orange-methyl green fluorescent staining of nucleoli. Stain Technol 56:343–348PubMedGoogle Scholar
  31. Gallagher TF (1983) Light stimulated transcription of genes for 2 chloroplast polypeptides in isolated pea leaf nuclei. Eur Mol Biol Organ J 1:1493–1498Google Scholar
  32. Garland PB, Mathias AP (1977) Biochemistry of the cell nucleus. The Biochemical Society, LondonGoogle Scholar
  33. Gealt MA, Sheir-Neiss G, Morris NR (1976) The isolation of nuclei from the filamentous fungus Aspergillus nidulans J Gen Microbiol 94:204–210PubMedGoogle Scholar
  34. Goessens G (1984) Nucleolar structure. Int Rev Cytol 87:107–158PubMedCrossRefGoogle Scholar
  35. Grass-Bellard M, Oudet P, Chambon P (1973) Isolation of high molecular-weight DNA from mammalian cells. Eur J Biochem 36:32–38CrossRefGoogle Scholar
  36. Gregor D, Reinert J, Matsumoto H (1974) Changes in chromosomal proteins from embryo induced carrot cells. Plant Cell Physiol 15:875–881Google Scholar
  37. Gupta DNS, Sen SP (1982) Phytochrome regulation of uptake of metabolites by coconut nuclei in vitro. Plant Sci Lett 24:61–66CrossRefGoogle Scholar
  38. Hadlaczky G, Bioztray G, Praznovszky T, Dudits D (1983) Mass isolation of plant chromosomes and nuclei. Planta 157:278–285CrossRefGoogle Scholar
  39. Hamilton VT, Habberset MC, Herman CJ (1980) Flow microfluorometric analysis of cellular DNA: critical comparison of mithramycin and propidium iodide. J Histochem Cytochem 28:1125–1128PubMedCrossRefGoogle Scholar
  40. Harland J, Jackson JF, Yeoman MM (1973) Changes in some enzymes involved in DNA biosynthesis following induction of division in cultured plant cells. J Cell Sci 13:121–138PubMedGoogle Scholar
  41. Hogeboom GH, Schneider WC (1952) Cytochemical studies VI. Synthesis of diphospho-pyridine nucleotide by liver cell nuclei. J Èiol Chem 197:611–620Google Scholar
  42. Hughes BG, Hess WM, Smith MA (1977) Ultrastructure of nuclei isolated from plant protoplasts. Protoplasma 93:267–274CrossRefGoogle Scholar
  43. Hull HM, Howshaw RW, Wang J-Chyong (1982) Cytofluorometric determination of nuclear DNA in living and preserved algae. Stain Technol 57:273–282PubMedGoogle Scholar
  44. Ide GJ (1981) Nucleoside 5’-y-S-triphosphates will initiate transcription in isolated yeast nuclei. Biochemistry 20:2633–2638PubMedCrossRefGoogle Scholar
  45. Jackson JF, Linskens HF (1978) Evidence for DNA repair after ultraviolet irradiation of Petunia hybrida pollen. Mol Gen Genet 161:117–120Google Scholar
  46. Kao KN (1975) A nuclear staining method for plant protoplasts. In: Gamborg OL, Wetter LR (eds) Plant-tissue culture methods. Natl Res Counc Can, Saskatoon, pp 60–61Google Scholar
  47. Kasten FH (1960) The chemistry of Schiffs reagent. Int Rev Cytol 10:1–100Google Scholar
  48. Kasten FH (1964) The Feulgen reaction, an enigma in cytochemistry. Acta Histochem 17:88–89PubMedGoogle Scholar
  49. Kohen E, Siebert G, Kohen C (1971) Transfer of metabolites across nuclear membranes.Hoppe-Seyler’s Z Physiol Chem 352:927–937PubMedCrossRefGoogle Scholar
  50. Kuehl L (1964) Isolation of plant nuclei. Z Naturforsch 19B:525–532Google Scholar
  51. Korson R (1951) A differential stain for nucleic acids. Stain Technol 26:265–270PubMedGoogle Scholar
  52. Kurnick NB (1955) Pyronin Y in the methyl green-pyronin histological stain. Stain Technol 30:213–230PubMedGoogle Scholar
  53. Kuroiwa T (1976) Isolation of mitochondrial nuclei and their fine structures. J Electron Microsc 25:182Google Scholar
  54. Kuroiwa T (1982) Mitochondrial nuclei. Int Rev Cytol 75:1–60PubMedCrossRefGoogle Scholar
  55. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  56. LaLoue M, Courtois D, Manigault P (1980) Convenient and rapid fluorescent staining of plant cell nuclei with “33258” Hoechst. Plant Sci Lett 17:175–179CrossRefGoogle Scholar
  57. Leuchtenberger C (1958) Quantitative determination of DNA in cells by Feulgen micro- spectrophotometry. In: Danielli JE (ed) General cytochemical methods, vol 1. Academic Press, New York, pp 219–278Google Scholar
  58. Lohr D, Kovaic RT, Van Holde KE (1977) Quantitative analysis of the digestion of yeast chromatin by Staphylococcal nuclease. Biochemistry 16:463–471PubMedCrossRefGoogle Scholar
  59. Lynch MJ, Buetow DE (1975) Isolation of intact nuclei from Euglena gracilis Exp Cell Res 91:344–348PubMedCrossRefGoogle Scholar
  60. MacPherson P, Fillon WG (1981) Karyotype analyses and the distribution of constitutive heterochromatin in five species of Pinus J Hered 72:193–198Google Scholar
  61. Malmberg RL, Griesbach RJ (1983) Chromosomes from protoplasts - isolation, fractionation and uptake. In: Hollaender, A (ed) Genetic engineering of plants, vol 26. Basic life sciences. Plenum, New York, pp 195–201Google Scholar
  62. Manne K, Mecke D (1980) Isolation and characterization of nuclei and nuclear membranes from Saccharomyces cerevisiae protoplasts. FEBS Lett 122:95–99CrossRefGoogle Scholar
  63. Mascarenhas JP, Berman-Kurtz M, Kulikowski RR (1974) Isolation of plant nuclei. Methods Enzymol 31:558–564PubMedCrossRefGoogle Scholar
  64. Matsumoto H, Gregor D, Reinert J (1976) Changes in chromatin of Daucus carota cells during embryogenesis. Phytochemistry 14:41–47CrossRefGoogle Scholar
  65. Matthysse AG, Rich K, Kontak C (1975) RNA synthesis in crown gall tumour cells. Plant Physiol 56:24Google Scholar
  66. May R (1980) Cytochemical characterization of spindle pole bodies of isolates nuclei from the yeastKluyveromyces fragils by digestion with nucleases. Protoplasma 102:21–30CrossRefGoogle Scholar
  67. Mazliak P, Robert D, Decotte-Justin AM (1977) Lipid metabolism in isolated nuclei from germinating sunflower hypocotyls. Plant Sci Lett 9:211–234CrossRefGoogle Scholar
  68. McClintock B (1934) The relation of a particular chromosomal element to the development of the nucleoli in Zea mays Z Zellforsch Mikrosk Anat 21:294–328CrossRefGoogle Scholar
  69. Mennes AM, Bouman H, van der Burg MPH, Liebenga KR (1978) RNA synthesis in isolated tobacco callus nuclei and the influence of phytohormones. Plant Sci Lett 13:329–339CrossRefGoogle Scholar
  70. Mennes AMO, Voogt E, Libbenga KR (1977) The isolation of nuclei from cultured tobacco pith explants. Plant Sci Lett 8:171–177CrossRefGoogle Scholar
  71. Mendelsohn ML (1958) The two wavelength method of microspectrophotometry II. A set of tables to facilitate the calculations. J Biophys Biochem Cytol 4:415–424PubMedCrossRefGoogle Scholar
  72. Mepham RH, Lane GR (1969) Nucleopores and polyribosome formation. Nature 221:288–289CrossRefGoogle Scholar
  73. Miksche JP (1966) DNA synthesis in primary roots of Glycine max during germination. Can J Bot 46:115–120Google Scholar
  74. Miyakawa I, Aoi H, Sands N, Kuroiwa T (1984) Fluorescence microscopic studies of mitochondrial nucleoids during mieosis and sporulation in the yeast, Saccharomyces cererisiae J Cell Sci 66:21–38PubMedGoogle Scholar
  75. Murata M (1983) Staining air dried protoplasts for study of plant chromsomes. Stain Tech- nol 58:101–106Google Scholar
  76. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  77. Nagata T, Ishii S (1979) A rapid method for isolation of mesophyll protoplasts. Can J Bot 57:1820–1823CrossRefGoogle Scholar
  78. Nagl W (1981) Polytene chromosomes of plants. Int Rev Cytol 73:21–54CrossRefGoogle Scholar
  79. Nevins JR (1983) The pathway of eukaryotic mRNA formation. Annu Rev Biochem 52:441–466PubMedCrossRefGoogle Scholar
  80. Palit S, Dutta R, Sarkar G, Dube DK (1982) Nucleic acid synthesis by isolated plant nuclei. Indian J Biochem Biophys 19:91–94PubMedGoogle Scholar
  81. Parish RW, Schmidlin S, Fuhrer S, Widmer S (1980) Electrophoretic isolation of nucleosomes from Dictyostelium descoideum nuclei and nucleoli proteins associated with monomers and dimers. FEBS Lett 110:236–240PubMedCrossRefGoogle Scholar
  82. Preiss J, Handler P (1958) Biosynthesis of diphosphopyridine nucleotide. J Biol Chem 233:493–500PubMedGoogle Scholar
  83. Price CA (1974) Plant cell fractionation. Methods Enzymol 31:501–519PubMedCrossRefGoogle Scholar
  84. Rho JH, Chipchase MI (1962) Incorporation of tritiated cytidine into RNA by isolated pea nuclei. J Cell Biol 14:183–192PubMedCrossRefGoogle Scholar
  85. Roman R, Caboche M, Lark KG (1980) Replication of DNA by nuclei isolated from soybean suspension cultures. Plant Physiol 66:726–730PubMedCrossRefGoogle Scholar
  86. Roque AL, Jafarey NA, Coulter P (1965) A stain for the histochemical demonstration of nucleic acids. Exp Mol Pathol 4:266–274CrossRefGoogle Scholar
  87. Ruch F (1973) Quantitative determination of DNA and protein in single cells. In: Thaer AA, Sernetz M (eds) Fluorescence techniques in cell biology. Springer, Berlin Heidelberg New York, pp 89–93Google Scholar
  88. Sau H, Sharma AK, Choudhuri RK (1980) DNA, RNA and protein content of isolated nuclei from different plant organs. Indian J Exp Biol 18:1519–1522Google Scholar
  89. Schweiger H-G, Berger S (1979) Nucleocytoplasmic interrelationships inAcetabularia and some other Dasycladaceae. Int Rev Cytol [Suppl] 9:12–44Google Scholar
  90. Scott NS, Possingham JV (1980) Chloroplast DNA in expanding spinach leaves. J Exp Bot 31:1081–1092CrossRefGoogle Scholar
  91. Scott NS, Timmis JN (1984) Homologies between nuclear and plastid DNA in spinach. Theor Appl Genet 67:279–288CrossRefGoogle Scholar
  92. Sheridan WF (1973) Nonaqueous isolation of nuclei from Lily pollen and an examination of their histones. Z Pflanzenphysiol 68:450–459Google Scholar
  93. Sheridan WF (1978) Preservation of synaptonemal complex structure in unfixed meiotic cells and isolated nuclei of Lilium. J Cell Biol 79:185AGoogle Scholar
  94. Simon JH, Becker WM (1976) A polyethylene glycol/dextran procedure for the isolation of chromatin proteins (histones and nonhistones) from wheat germ. Biochim Biophys Acta 454:154–171PubMedGoogle Scholar
  95. Smetana K, Busch H (1966) Studies on staining and localization of acidic nuclear proteins in the walker 256 carcinosarcoma. Cancer Res 26:331–337PubMedGoogle Scholar
  96. Smith J, Keir HM (1963) DNA nucleotidyltransferase in nuclei and cytoplasm prepared from thymus tissue in non-aqueous media. Biochim Biophys Acta 68:578–588CrossRefGoogle Scholar
  97. Spicer SS (1961) Differentiation of nucleic acids by staining at controlled pH and by Schiff-methylene blue sequence. Stain Technol 36:337–340PubMedGoogle Scholar
  98. Spiker S, Murray MG, Thompson WF (1983) DNase I sensitivity of transcriptionally active genes in intact nuclei and isolated chromatin of plants. Proc Nat Acad Sci USA 80:815–819PubMedCrossRefGoogle Scholar
  99. Takats ST, Wever GH (1972) DNA polymerase and DNA nuclease activities in S-compe- tent and S-incompetent nuclei from Tradescantia pollen grains. Exp Cell Res 69:25–28CrossRefGoogle Scholar
  100. Talbert KJ, Russell PJ (1982) Nuclear buoyant density determination and the purification and characterization of wild typeNeurospora crass a using percoll density gradients. Plant Physiol 70:704–708CrossRefGoogle Scholar
  101. Tallman G, Reeck GP (1980) Isolation of nuclei from plant protoplasts without the use of a detergent. FEBS Lett 18:271–276Google Scholar
  102. Vlasak J (1981) Effect of different disintegration techniques and media on yield and appearance of isolated nuclei. Biologia Plant 23:406–413CrossRefGoogle Scholar
  103. Ward DC, Reich E, Goldberg IH (1965) Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149:1259–1263PubMedCrossRefGoogle Scholar
  104. Werker E, Lerner HR, Weinberg R, Poljajoff-Mayber A (1983) Structural changes occurring in nuclei of barley root cells in response to a combined effect of salinity and ageing. Am J Bot 70:222–225CrossRefGoogle Scholar
  105. Willmitzer L, Wagner KG (1981) The isolation of nuclei from tissue cultured plant cells. Exp Cell Res 135:69–78PubMedCrossRefGoogle Scholar
  106. Willmitzer L, De Beuckeleer M, Lemmers M, Van Montagu M, Schell J (1980) DNA from Ti plasmid present in nucleus and absent from plastids of crown gall plant cells. Nature 287:359–361CrossRefGoogle Scholar
  107. Yamaguchi H, Naito T, Tatara A (1977) Purification of DNA polymerase isolated from the nuclei in dry barley seed embryo. J Radiat Res 18:31CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1985

Authors and Affiliations

  • J. F. Jackson

There are no affiliations available

Personalised recommendations