Subnuclear Components. Preparation and Fractionation

334 Pages·1976·7.285 MB·English

by  G. D. Birnie (Eds.)

SUBNUCLEAR COMPONENTS
Preparation
and Fractionation
Edited by
G. D. BIRNIE Ph.D.
Senior Scientist, Beatson Institute for Cancer Research, Glasgow
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First published 1976
© Butterworth & Co (Publishers) Ltd 1976
ISBN 0 408 70729 1 c \ v \y \
Library of Congress Cataloging in Publication Data
Main entry under title:
Subnuclear components.
Includes bibliographies and index.
1. Cell nuclei. 2. Cell fractionation. 3. Cell
organelles. I. Birnie, G. D.
QH595.S76 574.8'732 75-32547
ISBN 0-408-70729-1
Set by Cold Composition Ltd, Tonbridge, Kent
Printed in England by J. W. Arrowsmith Ltd, Bristol
PREFACE
To all intents and purposes this book is a sequel to Subcellular
Components: Preparation and Fractionation*. Just as the latter
subjected the many techniques then available for dissecting the
eucaryotic cell to a detailed, critical examination so this book attempts
to expose the methodology for isolating subnuclear components to a
similar kind of assessment. Thus, although it contains detailed
descriptions of many methods for isolating the constituents of nuclei,
this book is not another in a series of review articles, nor yet just a
collection of favourite methods. Its purpose is to provide a critical
examination and comparison of the methods currently available for
isolating the major components of nuclei, to draw attention to the
sources and consequences of difficulties which are often unsuspected or
ignored, and to suggest criteria which can be used to assess the
biochemical and biological quality of the preparations.
The book opens with a detailed discussion of methods for isolating
nuclei for the simple reason that the first step in isolating most nuclear
components is the preparation of clean, undegraded nuclei. The main
emphasis of this chapter lies in a discussion of the variation in
technique required when dealing with different types of cells and
tissues; it is, therefore, complementary to the chapter on nuclear
isolation methods in Subcellular Components, not a replacement for it.
The other chapters are devoted to discussions of the methods for
isolating each of the major components of the nucleus — nuclear
membranes, nucleoli, chromatin, chromatin proteins, nuclear RNA and
DNA. In order to achieve the stated purpose of the book, each
contributor has included: a discussion of the general type of methodo­
logy involved; critical comparisons of the more important methods
which are being, or have been, used; detailed descriptions of methods
which have been found to be particularly useful; a discussion of criteria
* Second edition edited by G. D. Birnie, published by Butterworths, London and
University Park Press, Baltimore, 1972.
which can be used to determine the extent to which the preparation is
degraded and the degree to which it is contaminated with cytoplasmic,
or other nuclear, components; and comments on the effects which
degradation or contamination might have on any assessment of the
biochemical and biological activity of a nuclear component. One
feature of each contribution which it is hoped will be of particular use
is the explanations of the 'why' as well as the 'how' of procedures; this
should enable readers to assess where it might be possible to vary a
method to suit a particular purpose.
Although many methods are described in greater or lesser detail in
this book, a large number have been omitted. These omissions are
deliberate, for no book with the aim of this one could hope to be
wholly comprehensive and remain readable and useful in the way this
book is intended to be. The opinions expressed in this book necessarily
represent the personal views of the authors. However, it should be
emphasized that the authors are all biologists with much practical
experience of their topics; moreover, in a number of instances they
took the time and trouble to run through procedures with which they
had had no previous personal experience in order to be able to give a
properly balanced assessment of their usefulness. I wish, therefore, to
record my gratitude to them, not only for their ready and willing
co-operation in the production of this book, but also for the care and
effort which they put into the preparation of their contributions. I
would also like to thank the authors and the publishers for the patience
they showed when the preparation of the book fell behind schedule,
and Mrs Rae Fergusson for her skilled and invaluable secretarial
assistance.
G. D. BIRNIE
CONTRIBUTORS
Anne M. Baker
Department of Zoology, University of Edinburgh, West Mains Road,
Edinburgh, EH9 3JT
G. D. Birnie
Beatson Institute for Cancer Research, 132 Hill Street, Glasgow, G3
6UD
M. E. Bramwell
Sir William Dunn School of Pathology, University of Oxford, South
Parks Road, Oxford, 0X1 3RE
Peter H. W. Butterworth
Department of Biochemistry, University College London, Gower Street,
London, WC1E 6BT
D. J. Fry
Department of Anatomy, Medical Sciences Institute, University of
Dundee, Hawkhill, Dundee, DD1 4HN
E. W. Johns
Chester Beatty Research Institute, Institute of Cancer Research: Royal
Cancer Hospital, Fulham Road, London, SW3 6JB
Marlene Koplitz
Department of Pathology, School of Medicine, University of Washing
ton, Seattle, Washington 98195, U.S.A.
Ulrich E. Loening
Department of Zoology, University of Edinburgh, West Mains Road,
Edinburgh, EH9 3JT
A. J. MacGillivray
Beatson Institute for Cancer Research, 132 Hill Street, Glasgow, G3
6UD
D. Rickwood
Beatson Institute for Cancer Research, 132 Hill Street, Glasgow, G3
6UD
Douglas E. Smuckler
Department of Pathology, School of Medicine, University of Washing
ton, Seattle, Washington 98195, U.S.A.
Edward A. Smuckler
Department of Pathology, School of Medicine, University of Washing
ton, Seattle, Washington 98195, U.S.A.
1
ISOLATION OF ANIMAL CELL NUCLEI
Edward A. Smuckler, Marlene Koplitz and Douglas E. Smuckler
Department of Pathology, School of Medicine, University of Washington, Seattle
Cell nuclei were identified in 1830 by Robert Brown. Their geo­
graphical position roughly in the centre of the cell, and the significance
of their several morphological features as diagnostic criteria in disease
states, augmented interest in their biological role. The discovery that
the nucleus contains DNA, and that RNA is synthesized there and
transported from the nucleus to the cytoplasm, further stimulated an
analysis of its structure and function. Identification of an informational
coding system within the nucleus, and of a means of translation of this
coding system for regulating cell functions, suggested a biochemical
structure-function relationship, but left unanswered questions regarding
specific morphological relationships for these activities. A brief reflec­
tion on the premise on which the messenger concept and the operon
hypothesis are based reveals that it involves naked DNA with no barrier
to transmission of either activators or repressors to and from the
genetic code, nor barriers to the transport of transcribed messengers to
their functional sites. Eucaryotes have proteins associated with their
DNA, and a membrane barrier separates the resulting complex from the
rest of the cytoplasm. In fact, this physical separation constitutes what
we recognize as the nucleus, and it causes some of the problems
involved in elucidating the structural and functional roles of the
component parts of the nucleus. Elucidation of these roles may be
achieved in part by physically separating the several nuclear com­
ponents. Many techniques for doing this require the isolation of nuclei
as a first step. A better basis for understanding structural relationships
within, and for analysis of, isolated nuclei will be achieved if some
knowledge of nuclear ultrastructure is available.
1
2 ISOLATION OF ANIMAL CELL NUCLEI
THE STRUCTURE AND FUNCTION OF NUCLEI
NUCLEAR STRUCTURE
There is significant similarity in the structure of the nucleus in different
species of eucaryotes, and in the several tissues constituting these
organs or organisms. One specific significant structural feature dif­
ferentiating the eucaryote from the procaryote is that the nucleus in
eucaryotic cells is surrounded by a nuclear envelope (Figures 1 and 2).
The nuclear envelope is continuous with the endoplasmic reticulum,
probably in all cells (Figure 2, 2 and 3a). The ergastoplasm provides a
reflection over the surface of the nucleus, forming an outer leaflet
(frequently studded with ribosomes) which is then reflected onto the
surface of the chromatin net forming an inner leaflet of the same
membrane. A real space exists between these leaflets, and is continuous
with the cisternae of the endoplasmic reticulum (Figure 2). The points
of reflection at which the envelope folds back on itself form nuclear
pores. These pores are not empty spaces, but are considered to have a
fibrillar ground substance present within them (Figures I and 3a).
Indeed there seems to be some type of central channel, and heavy metal
transport has been shown to occur through these entities.
The inner leaflet nuclear membrane is a less defined structure, from
a morphological standpoint. The assumption that it represents a
continuation of the endoplasmic reticulum is derived entirely from
morphological grounds. However, even nuclei isolated by methods
utilizing detergents (see p. 19), which ostensibly dissolve the nuclear
envelope, still have some type of retaining barrier. This inner barrier
may consist of two components, the inner leaflet of the envelope and
some form of condensation of the nuclear material. Note that, in Figure
2, an inner condensation of membrane is visible and there appears to be
an orderly array of chromatin granules juxtaposed to this surface.
Figures 4 and 5 show this barrier effect quite clearly; it is also evident
in Figures 9 and 10 (pp. 14 and 15), especially the latter, in which
retention of virus particles in detergent-washed nuclei is shown. The
nature of this barrier, and its identification with the nuclear envelope, is
not yet certain. Details of the isolation of nuclear envelopes appear in
Chapter 2.
It is generally assumed that the nucleus is roughly spheroidal in
shape; however, nuclei show a remarkable plasticity and variability in
size and shape in several different tissues. The configuration of the
nucleus can be further distorted by the presence of invaginations of the
cytoplasm forming large pockets within the nuclear structure (Figure
7). This is specifically true of tumour tissue, and it also provides for
further difficulties in isolating nuclei (see p. 52). In addition, the
structure of the nuclear envelope, the size of the pores, and the
E. A. SMUCKLER, M. KOPLITZ AND D. E. SMUCKLER 3
distensions of the envelope itself are remarkably plastic in tumour
tissues. These factors must be recalled when considering the morpho­
logical appearance of nuclei isolated from these sources.
Chemically, the nucleus consists of a mixture of DNA, RNA and
nuclear-associated proteins, including histones and acidic proteins. The
morphological appearance of the nuclear structure depends in part
upon the means of fixation used, the physiological status of the tissue
before isolation of the nuclei, and the treatment of the nuclei before
fixation. In general, aldehyde fixatives are associated with an enhanced
clumping of nuclear material, particularly chromatin (Figure 5), whereas
the osmium-based fixatives are believed to result in a more physio­
logical dispersion of material within the nucleus.
The nucleoplasm in well fixed (osmium tetroxide) nuclei examined
by electron microscopy has several components. The chromatin
constitutes the largest recognizable mass, rather more uniformly
distributed in osmium-fixed material (Figure 1). These clumps consist
of a more or less densely packed granular-fibrillar mass. The more
densely packed material has been designated heterochromatin, the
looser material euchromatin (Frenster, Allfrey and Mirsky, 1963). Once
more, the relative aggregation of chromatin and the proportions of
condensed and extended material must be considered in the contexts of
cell physiology and methods of isolation, incubation and fixation. In
cells subject to anoxia, as well as in nuclei not fixed in osmium or won
by prolonged extraction, there is a clumping of the chromatin material,
which aligns itself along the nuclear membrane and, frequently, as large
masses within the nuclear substance (Figures 6 and 8). There is a
nucleolar-associated condensation of chromatin in normal cells, and
frequently this condensation will abut against the nuclear envelope in
active cells.
Among the chromatin is a space designated the interchromatinic
area which is relatively electron-lucent and apparently structureless. It
is believed to be composed of protein without a more defined structure.
Within this area, however, granular deposits are frequently observed,
and these have been designated interehromatin granules. They appear
singly, or as clusters, or irregularly arranged, are frequently rather
electron-dense, and have diameters between 20 and 25 nm. In other cell
types similar granules occur near the chromatin, and are called
perichromatin granules. They are somewhat larger (30—35 nm), and are
often surrounded by relatively electron-lucent areas forming halos. A
variety of other structures, including glycogen and virus-like particles,
can also be found within the nuclear structure. A detailed description
of these entities is beyond the scope of this particular review.
Also within the nucleus is the nucleolus (for a review of nucleolar
structure, see National Cancer Institute Monograph, 23, 1965). This
4 ISOLATION OF ANIMAL CELL NUCLEI
serpentine structure is composed of two morphologically separate
components, the nucleolonema and the pars amorpha. The latter is a
relatively electron-lucent area embedded within the serpentine nucleo-
lenema. The nucleolenema also has two components, a fibrillar one and
a granular one, the latter structure resembling ribosomes in size and
morphology. The nucleolus is particularly responsive to physiological
and pathological stimuli, showing significant morphological differences
during differentiation and regeneration, and under physiological and
pathological stress. Considerable enlargement, enhancement and separa­
tion of the several components of the nucleolenema have been
described.
These several configurations and the component parts of the nucleus
vary significantly from tissue to tissue, from organ to organ, and among
the phylogenetic groupings. For this reason it is important to identify
the 'normal' structure when comparisons are made with the isolated
material.
NUCLEAR FUNCTION
There is abundant evidence that the nucleus plays a significant role in
replication of DNA for mitosis, for non-mitotic DNA synthesis (in
repair) and in the formation of the several species of RNA. In addition
there is a variety of agencies that modify the RNA synthesized, and
regulate its transport to the cytoplasm. It has also been suggested that
the nucleus has autonomous capacity to form protein; indeed, it has
been suggested that several of the enzymes active in the nucleus may be
formed directly within the nuclear envelope. It has also been suggested
that the nucleus has autonomous powers of respiration and respiratory
control.
REASONS FOR ISOLATING NUCLEI
In this text it is redundant to identify in detail the many reasons for
isolating nuclei. It should be self-evident that detailed analysis of the
enzymology and the biological significance of several nuclear activities
can be discerned only by isolating the nucleus and examining its
components individually, and separate from whole cells. Not only is
there significance in ascertaining the biological function of the
components of the nucleus, but more recently it has become very clear
that a detailed examination of the mechanisms of abnormal function
and of pathological states must be done using isolated nuclei. Nowhere
is this more evident than in attempting to define the several roles of
mutagenesis, chemical carcinogenesis and acute toxicity. Furthermore,
strict analysis of the genetics of modifications of cells requires the
isolation of the replicating species of DNA. Inherent in these studies is
the desire to mimic in a more controlled environment some of the