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The Nucleus — Cytological Methods and Isolation for Biochemical Studies

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Cell Components

Part of the book series: Modern Methods of Plant Analysis ((MOLMETHPLANT,volume 1))

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Abstract

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.

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References

  • Ansa OA, Bowyer JW, Shepherd RJ (1982) Evidence for replication of cauliflower mosaicvirus DNA in plant nuclei. Virology 121:147–156

    Article  PubMed  CAS  Google Scholar 

  • 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 26:31–38

    Article  Google Scholar 

  • Armstrong JA (1956) Histochemical differentiation of nucleic acids by means of induced fluorescence. Exp Cell Res 11:640–643

    Article  PubMed  CAS  Google Scholar 

  • Atkinson MR, Jackson JF, Morton RK (1961) Nicotinamide mononucleotide adenylyl transferase of pig liver nuclei. Biochem J 80:318–323

    PubMed  CAS  Google Scholar 

  • Belling J (1921) On counting chromosomes in pollen-mother cells. Am Nat 55:573–574

    Article  Google Scholar 

  • Bennet MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Philos Trans R Soc Lond B 274:227–274

    Article  Google Scholar 

  • 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 York

    Google Scholar 

  • Bonner J (1976) The Nucleus. In: Bonner J, Varner JE (eds) Plant biochemistry, 3rd edn. Academic Press, New York, pp 37–64

    Google Scholar 

  • 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 12:3–65

    Article  CAS  Google Scholar 

  • 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 22:361–368

    Article  CAS  Google Scholar 

  • 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 63:640–646

    PubMed  CAS  Google Scholar 

  • Brewer EN (1979) Repair of radiation-induced DNA double-strand breaks in isolated nuclei of Physarum polycephalum Radiat Res 79:368–376

    Article  PubMed  CAS  Google Scholar 

  • Brightwell MD, Leech CE, O’Farrell MK, Whish WJ, Shall S (1975) Poly ADP ribose polymerase in Polysarum polycephalum Biochem J 147:119–130

    PubMed  CAS  Google Scholar 

  • Buetow DE (1976) Isolation of nuclei from protozoa and algae. In: Prescott DM (ed) Methods in cell biology. Academic Press, New York, pp 283–311

    Google Scholar 

  • Busch H, Smetana K (1970) The Nucleolus. Academic Press, New York, pp 548–575

    Google Scholar 

  • 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 78:1731–1735

    Article  PubMed  CAS  Google Scholar 

  • Capesius I, Meyer Y (1977) Isolation of nuclei from protoplasts of orchids. Cytobiologie 15:485–490

    Google Scholar 

  • Chabot JF, Leopold AC (1982) Ultrastructural changes of membranes with hydration in soybean seeds. Am J Bot 69:623–633

    Article  Google Scholar 

  • Chauveau J, Moulé Y, Rouiller CH (1956) Isolation of pure and unaltered liver nuclei. Morphology and biochemical composition. Exp Cell Res 11:317–321

    Google Scholar 

  • 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 73:746–753

    Article  PubMed  CAS  Google Scholar 

  • Coleman AW (1978) Visualization of chloroplast DNA with two fluorochromes. Exp Cell Res 114:95–100

    Article  PubMed  CAS  Google Scholar 

  • Dawson JRY, Thomas K, CI egg MT (1982) Purification of total cellular DNA from a single plant. Biochem Genet 20:209–219

    Article  Google Scholar 

  • Dhar AC, Shah CK (1982) Cytochemical method to localize acid nuclear proteins. Stain Technol 57:151–155

    PubMed  CAS  Google Scholar 

  • Dhillon SS, Berlyn GP, Miksche JP (1977) Requirement of an internal standard for micro-spectrophotometric measurement of DNA. Am J Bot 64:117–121

    Article  CAS  Google Scholar 

  • Dhillon SS, Berlyn GP, Miksche JP (1978) Nuclear DNA content in populations of Pinusrigida Am J Bot 65:192–196

    Article  CAS  Google Scholar 

  • Dounce AL (1943) Enzyme studies on isolated cell nuclei of rat liver. J Biol Chem 147:685–698

    CAS  Google Scholar 

  • 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 33:629–642

    Article  PubMed  CAS  Google Scholar 

  • 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 137:245–248

    Article  PubMed  CAS  Google Scholar 

  • Franke WW (1974) Structure, biochemistry, and functions of the nuclear envelope. Int Rev Cytol [Suppl] 4:72–236

    Google Scholar 

  • Franklin AL, Filion WG (1981) Acridine orange-methyl green fluorescent staining of nucleoli. Stain Technol 56:343–348

    PubMed  CAS  Google Scholar 

  • Gallagher TF (1983) Light stimulated transcription of genes for 2 chloroplast polypeptides in isolated pea leaf nuclei. Eur Mol Biol Organ J 1:1493–1498

    Google Scholar 

  • Garland PB, Mathias AP (1977) Biochemistry of the cell nucleus. The Biochemical Society, London

    Google Scholar 

  • Gealt MA, Sheir-Neiss G, Morris NR (1976) The isolation of nuclei from the filamentous fungus Aspergillus nidulans J Gen Microbiol 94:204–210

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Grass-Bellard M, Oudet P, Chambon P (1973) Isolation of high molecular-weight DNA from mammalian cells. Eur J Biochem 36:32–38

    Article  Google Scholar 

  • Gregor D, Reinert J, Matsumoto H (1974) Changes in chromosomal proteins from embryo induced carrot cells. Plant Cell Physiol 15:875–881

    CAS  Google Scholar 

  • Gupta DNS, Sen SP (1982) Phytochrome regulation of uptake of metabolites by coconut nuclei in vitro. Plant Sci Lett 24:61–66

    Article  Google Scholar 

  • Hadlaczky G, Bioztray G, Praznovszky T, Dudits D (1983) Mass isolation of plant chromosomes and nuclei. Planta 157:278–285

    Article  CAS  Google Scholar 

  • Hamilton VT, Habberset MC, Herman CJ (1980) Flow microfluorometric analysis of cellular DNA: critical comparison of mithramycin and propidium iodide. J Histochem Cytochem 28:1125–1128

    Article  PubMed  CAS  Google Scholar 

  • 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 13:121–138

    PubMed  CAS  Google Scholar 

  • Hogeboom GH, Schneider WC (1952) Cytochemical studies VI. Synthesis of diphospho-pyridine nucleotide by liver cell nuclei. J Èiol Chem 197:611–620

    CAS  Google Scholar 

  • Hughes BG, Hess WM, Smith MA (1977) Ultrastructure of nuclei isolated from plant protoplasts. Protoplasma 93:267–274

    Article  Google Scholar 

  • Hull HM, Howshaw RW, Wang J-Chyong (1982) Cytofluorometric determination of nuclear DNA in living and preserved algae. Stain Technol 57:273–282

    PubMed  CAS  Google Scholar 

  • Ide GJ (1981) Nucleoside 5’-y-S-triphosphates will initiate transcription in isolated yeast nuclei. Biochemistry 20:2633–2638

    Article  PubMed  CAS  Google Scholar 

  • Jackson JF, Linskens HF (1978) Evidence for DNA repair after ultraviolet irradiation of Petunia hybrida pollen. Mol Gen Genet 161:117–120

    CAS  Google Scholar 

  • 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 60–61

    Google Scholar 

  • Kasten FH (1960) The chemistry of Schiffs reagent. Int Rev Cytol 10:1–100

    CAS  Google Scholar 

  • Kasten FH (1964) The Feulgen reaction, an enigma in cytochemistry. Acta Histochem 17:88–89

    PubMed  CAS  Google Scholar 

  • Kohen E, Siebert G, Kohen C (1971) Transfer of metabolites across nuclear membranes.Hoppe-Seyler’s Z Physiol Chem 352:927–937

    Article  PubMed  CAS  Google Scholar 

  • Kuehl L (1964) Isolation of plant nuclei. Z Naturforsch 19B:525–532

    Google Scholar 

  • Korson R (1951) A differential stain for nucleic acids. Stain Technol 26:265–270

    PubMed  CAS  Google Scholar 

  • Kurnick NB (1955) Pyronin Y in the methyl green-pyronin histological stain. Stain Technol 30:213–230

    PubMed  CAS  Google Scholar 

  • Kuroiwa T (1976) Isolation of mitochondrial nuclei and their fine structures. J Electron Microsc 25:182

    Google Scholar 

  • Kuroiwa T (1982) Mitochondrial nuclei. Int Rev Cytol 75:1–60

    Article  PubMed  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • LaLoue M, Courtois D, Manigault P (1980) Convenient and rapid fluorescent staining of plant cell nuclei with “33258” Hoechst. Plant Sci Lett 17:175–179

    Article  CAS  Google Scholar 

  • 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 219–278

    Google Scholar 

  • Lohr D, Kovaic RT, Van Holde KE (1977) Quantitative analysis of the digestion of yeast chromatin by Staphylococcal nuclease. Biochemistry 16:463–471

    Article  PubMed  CAS  Google Scholar 

  • Lynch MJ, Buetow DE (1975) Isolation of intact nuclei from Euglena gracilis Exp Cell Res 91:344–348

    Article  PubMed  CAS  Google Scholar 

  • MacPherson P, Fillon WG (1981) Karyotype analyses and the distribution of constitutive heterochromatin in five species of Pinus J Hered 72:193–198

    Google Scholar 

  • 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 195–201

    Google Scholar 

  • Manne K, Mecke D (1980) Isolation and characterization of nuclei and nuclear membranes from Saccharomyces cerevisiae protoplasts. FEBS Lett 122:95–99

    Article  Google Scholar 

  • Mascarenhas JP, Berman-Kurtz M, Kulikowski RR (1974) Isolation of plant nuclei. Methods Enzymol 31:558–564

    Article  PubMed  CAS  Google Scholar 

  • Matsumoto H, Gregor D, Reinert J (1976) Changes in chromatin of Daucus carota cells during embryogenesis. Phytochemistry 14:41–47

    Article  Google Scholar 

  • Matthysse AG, Rich K, Kontak C (1975) RNA synthesis in crown gall tumour cells. Plant Physiol 56:24

    Google Scholar 

  • May R (1980) Cytochemical characterization of spindle pole bodies of isolates nuclei from the yeastKluyveromyces fragils by digestion with nucleases. Protoplasma 102:21–30

    Article  CAS  Google Scholar 

  • Mazliak P, Robert D, Decotte-Justin AM (1977) Lipid metabolism in isolated nuclei from germinating sunflower hypocotyls. Plant Sci Lett 9:211–234

    Article  CAS  Google Scholar 

  • McClintock B (1934) The relation of a particular chromosomal element to the development of the nucleoli in Zea mays Z Zellforsch Mikrosk Anat 21:294–328

    Article  Google Scholar 

  • 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 13:329–339

    Article  CAS  Google Scholar 

  • Mennes AMO, Voogt E, Libbenga KR (1977) The isolation of nuclei from cultured tobacco pith explants. Plant Sci Lett 8:171–177

    Article  CAS  Google Scholar 

  • Mendelsohn ML (1958) The two wavelength method of microspectrophotometry II. A set of tables to facilitate the calculations. J Biophys Biochem Cytol 4:415–424

    Article  PubMed  CAS  Google Scholar 

  • Mepham RH, Lane GR (1969) Nucleopores and polyribosome formation. Nature 221:288–289

    Article  Google Scholar 

  • Miksche JP (1966) DNA synthesis in primary roots of Glycine max during germination. Can J Bot 46:115–120

    Google Scholar 

  • 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 66:21–38

    PubMed  CAS  Google Scholar 

  • Murata M (1983) Staining air dried protoplasts for study of plant chromsomes. Stain Tech- nol 58:101–106

    CAS  Google Scholar 

  • Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight DNA. Nucleic Acids Res 8:4321–4325

    Article  PubMed  CAS  Google Scholar 

  • Nagata T, Ishii S (1979) A rapid method for isolation of mesophyll protoplasts. Can J Bot 57:1820–1823

    Article  Google Scholar 

  • Nagl W (1981) Polytene chromosomes of plants. Int Rev Cytol 73:21–54

    Article  CAS  Google Scholar 

  • Nevins JR (1983) The pathway of eukaryotic mRNA formation. Annu Rev Biochem 52:441–466

    Article  PubMed  CAS  Google Scholar 

  • Palit S, Dutta R, Sarkar G, Dube DK (1982) Nucleic acid synthesis by isolated plant nuclei. Indian J Biochem Biophys 19:91–94

    PubMed  CAS  Google Scholar 

  • 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 110:236–240

    Article  PubMed  CAS  Google Scholar 

  • Preiss J, Handler P (1958) Biosynthesis of diphosphopyridine nucleotide. J Biol Chem 233:493–500

    PubMed  CAS  Google Scholar 

  • Price CA (1974) Plant cell fractionation. Methods Enzymol 31:501–519

    Article  PubMed  CAS  Google Scholar 

  • Rho JH, Chipchase MI (1962) Incorporation of tritiated cytidine into RNA by isolated pea nuclei. J Cell Biol 14:183–192

    Article  PubMed  CAS  Google Scholar 

  • Roman R, Caboche M, Lark KG (1980) Replication of DNA by nuclei isolated from soybean suspension cultures. Plant Physiol 66:726–730

    Article  PubMed  CAS  Google Scholar 

  • Roque AL, Jafarey NA, Coulter P (1965) A stain for the histochemical demonstration of nucleic acids. Exp Mol Pathol 4:266–274

    Article  CAS  Google Scholar 

  • 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 89–93

    Google Scholar 

  • Sau H, Sharma AK, Choudhuri RK (1980) DNA, RNA and protein content of isolated nuclei from different plant organs. Indian J Exp Biol 18:1519–1522

    Google Scholar 

  • Schweiger H-G, Berger S (1979) Nucleocytoplasmic interrelationships inAcetabularia and some other Dasycladaceae. Int Rev Cytol [Suppl] 9:12–44

    Google Scholar 

  • Scott NS, Possingham JV (1980) Chloroplast DNA in expanding spinach leaves. J Exp Bot 31:1081–1092

    Article  Google Scholar 

  • Scott NS, Timmis JN (1984) Homologies between nuclear and plastid DNA in spinach. Theor Appl Genet 67:279–288

    Article  CAS  Google Scholar 

  • Sheridan WF (1973) Nonaqueous isolation of nuclei from Lily pollen and an examination of their histones. Z Pflanzenphysiol 68:450–459

    Google Scholar 

  • Sheridan WF (1978) Preservation of synaptonemal complex structure in unfixed meiotic cells and isolated nuclei of Lilium. J Cell Biol 79:185A

    Google Scholar 

  • 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 454:154–171

    PubMed  CAS  Google Scholar 

  • Smetana K, Busch H (1966) Studies on staining and localization of acidic nuclear proteins in the walker 256 carcinosarcoma. Cancer Res 26:331–337

    PubMed  CAS  Google Scholar 

  • Smith J, Keir HM (1963) DNA nucleotidyltransferase in nuclei and cytoplasm prepared from thymus tissue in non-aqueous media. Biochim Biophys Acta 68:578–588

    Article  CAS  Google Scholar 

  • Spicer SS (1961) Differentiation of nucleic acids by staining at controlled pH and by Schiff-methylene blue sequence. Stain Technol 36:337–340

    PubMed  CAS  Google Scholar 

  • 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 80:815–819

    Article  PubMed  CAS  Google Scholar 

  • 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 69:25–28

    Article  Google Scholar 

  • 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 70:704–708

    Article  Google Scholar 

  • Tallman G, Reeck GP (1980) Isolation of nuclei from plant protoplasts without the use of a detergent. FEBS Lett 18:271–276

    CAS  Google Scholar 

  • Vlasak J (1981) Effect of different disintegration techniques and media on yield and appearance of isolated nuclei. Biologia Plant 23:406–413

    Article  Google Scholar 

  • Ward DC, Reich E, Goldberg IH (1965) Base specificity in the interaction of polynucleotides with antibiotic drugs. Science 149:1259–1263

    Article  PubMed  CAS  Google Scholar 

  • 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 70:222–225

    Article  Google Scholar 

  • Willmitzer L, Wagner KG (1981) The isolation of nuclei from tissue cultured plant cells. Exp Cell Res 135:69–78

    Article  PubMed  CAS  Google Scholar 

  • 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 287:359–361

    Article  CAS  Google Scholar 

  • Yamaguchi H, Naito T, Tatara A (1977) Purification of DNA polymerase isolated from the nuclei in dry barley seed embryo. J Radiat Res 18:31

    Article  Google Scholar 

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Jackson, J.F. (1985). The Nucleus — Cytological Methods and Isolation for Biochemical Studies. In: Linskens, HF., Jackson, J.F. (eds) Cell Components. Modern Methods of Plant Analysis, vol 1. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-82587-3_17

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  • DOI: https://doi.org/10.1007/978-3-642-82587-3_17

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