Subnuclear Components

Subnuclear Components

Preparation and Fractionation

1st Edition - January 1, 1976

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  • Editor: G. D. Birnie
  • eBook ISBN: 9781483103402

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Subnuclear Components: Preparation and Fractionation focuses on the isolation of subnuclear components of eukaryotic cells. The book first discusses the isolation of nuclear envelopes from whole tissues. Topics include nuclear envelope in situ; general consideration and evaluation of isolation methods; and nuclear envelope isolation methods. The text describes the isolation of nucleoli, including the isolation of nucleoli from oocytes, nuclei, and physarum, and isolation of plant nucleoli. The book focuses on the preparation, characterization, and fractionation of chromatin. Emphasis is on the preparation of chromatin from interphase and metaphase cells; contaminants of chromatin; characteristics of isolated chromatin; and dissociation and reassociation of chromatin. The text also discusses fractionation and isolation of histones, including the preparation of individual histone fractions and isolation and purification of the five main histone fractions of calf thymus during one preparation. The book also looks at the preparation and properties of chromatin non-histone proteins, including isolation of nuclear RNA and isolation of DNA from eukaryotic cells. The selection is a good source of data for readers interested in the isolation of subnuclear constituents of eukaryotic cells.

Table of Contents

  • 1 Isolation of Animal Cell Nuclei

    The Structure and Function of Nuclei

    Nuclear Structure

    Nuclear Function

    Reasons for Isolating Nuclei

    Problems Encountered when Isolating Nuclei

    Cellular Heterogeneity

    Cell Disruption

    Isolation Media

    Separation of Nuclei from the Brei

    Criteria of Purity

    Specific Methods for Different Tissues

    Lower Eucaryotes

    Invertebrates and Amphibia

    Tissue-Culture Cells

    Vertebrate Organs and Tissues

    Tumor Tissues



    2 Isolation of Nuclear Envelopes from Whole Tissue

    The Nuclear Envelope In Situ



    Chemical Composition

    General Consideration of Isolation Methods

    Tonicity of Isolation Media

    Ionic Composition of Isolation Media

    pH of Isolation Media


    Homogenization Techniques

    High-Density Sucrose Centrifugation

    Isolation of Nuclear Envelopes from Nuclei

    Nuclear Envelope Isolation Methods

    Evaluation of Isolation Methods

    Morphological Assessment


    Enzyme Activities

    Biochemical Assessment of Contamination



    3 Isolation of Nucleoli

    Isolation of Nucleoli from Oöcytes

    Isolation of Nucleoli from Nuclei

    Disrupting Nuclei by Deoxyribonuclease

    Disrupting Nuclei by Conication

    Disrupting Nuclei by Pressure Methods

    Isolation of Nuclei and Nucleoli in Citric Acid

    Isolation of Nucleoli by Other Methods

    Isolation of Nucleoli from Physarum

    Isolation of Plant Nucleoli



    4 Preparation, Characterization and Fractionation of Chromatin

    Preparation of Chromatin from Inter-phase Cells

    From Animal Tissues and Cells

    From Cells other than Animal Cells

    Preparation of Chromatin from Meta-phase Cells

    Isolation of Meta-phase Cells

    Isolation, Purification and Fractionation of Meta-phase Chromosomes

    Contaminants of Chromatin

    Contaminants of Cytoplasmic Origin

    Contaminants of Nuclear Origin

    Characteristics of Isolated Chromatin

    Chemical Composition

    Physicochemical Properties

    Template Activity

    Electron Microscopic Studies

    Immunological Properties

    Fractionation of Chromatin

    Features Distinguishing Active and Inactive Chromatin

    Fragmentation of Chromatin

    Characterization of Chromatin Fractions

    Separation of Chromatin Fractions


    Dissociation and Reassociation of Chromatin

    Partial Deproteinization




    5 Fractionation and Isolation of Histones

    Isolation and Purification of the Five Main Histone Fractions of Calf Thymus during One Preparation

    Details of the Procedure

    Comments on the Procedure

    Purification of Histone Fractions

    Gel Exclusion Chromatography for the Separation of All Five Histones

    Preparation of Individual Histone Fractions

    Preparation of Histone F1

    Preparation of Histone Fractions other than F1

    Preparation of the Chicken-Erythrocyte-Specific Histone F2C

    Preparation of Whole Unfractionated Histones from Calf Thymus

    Preparation of Histones from Tissues Other than Thymus

    Characterization of Products

    Storage of Histones


    6 Preparation and Properties of Chromatin Non-Histone Proteins

    Biological Relevance of Non-Histone Proteins

    Control of Gene Expression

    Structure of Chromatin

    Post-Synthetic Modifications

    Methods for Isolating Non-Histone Proteins

    Methods Applied to Dehistoned Chromatins

    Methods Applied to Dissociated Chromatins

    Selective Extraction Procedures

    Fractionation of Non-Histone Proteins

    Ion-Exchange Chromatography

    Iso-Electric Docusing

    Gel Filtration

    Characterization of Non-Histone Proteins


    Amino Acid Composition


    Factors Affecting the Composition of Non-Histone Proteins

    Methodological Artifacts

    Contamination by other Cell Constituents

    Preparation of Nuclei and Chromatin

    Methods Developed in the Author's Laboratory


    Fractionation of Chromatin

    One-Dimensional Gel Electrophoresis in Sodium Dodecyl Sulphate

    Two-Dimensional Gel Electrophoresis

    Fractionation of non-Histone Proteins on a Preparative Scale

    Notes on the Methods



    7 Isolation of Nuclear RNA

    Problems Encountered in Isolating Nuclear RNA





    Comparison of Methods

    Method for Isolating Nuclear RNA

    Details of Procedure

    Extraction of Total Cellular RNA

    Extraction of Nucleolar RNA

    Extraction of Low-Molecular-Weight Nuclear RNA

    Assessment of Nuclear RNA Preparations

    Amounts Obtained


    Degradation and Aggregation

    Fractionation of Nuclear RNA


    8 Isolation of DNA from Eucaryotic Cells

    Properties of DNA and Problems Associated with its Isolation

    How 'Big' is DNA?

    To what Extent is the DNA Duplex Intact

    Association of Protein and RNA with DNA

    DeoxyriboTiuclease Activity in Ribonuclease and Proteolytic Enzymes

    Criteria for the Selection of an Isolation Procedure

    Comparison of Procedures Described

    Methods of Assessing Size, Integrity and Purity of DNA

    Estimation of Molecular Weight

    Estimation of Integrity

    Estimation of DNA and Contaminants

    Isolation of DNA: Procedure I

    Description of Procedure I

    Analysis of Steps in Procedure I, and Possible Modifications

    Properties of Product of Procedure I

    Comments on Procedure I

    Isolation of DNA: Procedure II

    Description of Procedure II

    Analysis of Steps in Procedure II

    Properties of Product of Procedure II

    Isolation of DNA: Procedure III

    Description of Procedure III

    Analysis of Steps in Procedure III, and Properties of the Product

    General Conclusions



Product details

  • No. of pages: 348
  • Language: English
  • Copyright: © Butterworth-Heinemann 1976
  • Published: January 1, 1976
  • Imprint: Butterworth-Heinemann
  • eBook ISBN: 9781483103402

About the Editor

G. D. Birnie

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