J. Cell Sci. I, 331-35° (1966)
Printed in Great Britain
331
ELECTRON- AND LIGHT-MICROSCOPE
OBSERVATIONS ON THE SPLEEN OF THE
NEWT TRITURUS
CRISTATUS:
THE SURFACE TOPOGRAPHY OF THE
MITOTIC CHROMOSOMES
H. G. DAVIES AND J. TOOZE
Medical Research Council Biophysics Research Unit and
Department of Biophysics, King's College, London
SUMMARY
In polychromatophil erythroblasts, and in cells containing little or no haemoglobin, the
surfaces of the mitotic chromosomes frequently bear finger-like projections averaging about
o-i /* in diameter. Both the mitotic and interphase chromosomes in polychromatophil erythroblasts from some newts bear, in addition, extensive sheets of chromatin, about 315-465 A thick,
depending on fixation, bounded on each side by nuclear envelope-like fragments. The remarkably constant width of the sheets in all cells indicates that chromosomes contain a constructional unit of fixed dimensions. This unit may have general significance, since other
workers have found envelope-limited sheets of similar dimensions in one plant and several
animal species. An hypothesis concerning the construction of chromosomes is discussed. It
seeks to relate the thickness of the sheets of chromatin to the average diameter of the majority
of the finger-like processes.
The large intranuclear granules, 400 A in diameter, in polychromatophil erythroblasts
survive in polysome-like configurations in dividing cells.
INTRODUCTION
The conclusion that chromosomes' possess a complex and definite internal organization' put forward by Wilson (1925) was based on evidence from genetic experiments
and observations, with the light microscope, of the morphology of chromosomes and
their precise behaviour during mitosis and meiosis. The genetic information is now
known to reside in the deoxyribose nucleic acid (DNA) molecule, but the organization
of DNA, protein and other molecules in the chromosome has still not been elucidated,
although new techniques and much pertinent information relating to chromosome
structure has accumulated since the publication of the third edition of Wilson's book.
In bacteriophage, bacteria and dinoflagellates the data have been reviewed by Kellenberger (1965), and in higher organisms by Gall (1958), Steffenson (1959), Kaufmann,
Gay & McDonald (i960), Callan & Lloyd (i960), Ris (1961), Swift (1962a), Taylor
(1962, 1963), Ris & Chandler (1963), and Beerman (1965). The data are derived from
chemical analysis (Allfrey & Mirsky, 1964; Swift, 1964; Rudkin, 1965), autoradiography (Taylor, 1959; Gall & Callan, 1962; Izawa, Allfrey & Mirsky 1963; Prescott,
1963), genetics (see Taylor (1963) for a discussion of reciprocal and non-reciprocal
332
H. G. Davies and J. Tooze
recombination), structural analysis by X-ray diffraction (Wilkins, 1956; Luzatti &
Nicolaieff, 1963) and light- and electron-microscopy (see reviews).
When sections, 300-1000 A in thickness, of chromosomes from dividing and interphase cells are examined in the electron microscope, threads and granules are observed,
but these display no specific arrangement or order and there is wide variation in their
size. The reported diameters range from 25 A in chromosomes from the salivary
gland of Drosopkila (Goodman & Spiro, 1962), 50-75 A in interphase nuclei of cells
from salamander (Hay & Revel, 1963), to 160 A in isolated nuclei from pea (Hyde,
1964). In extensive investigations, Ris & Chandler (1963) report as the basic unit a
thread 100 A in diameter, often associated in twos or fours to form thicker fibres,
each 100-A thread being composed of two 40-A threads. Apart from possible variation
between species and differing physiological conditions, the observed spatial arrangement of DNA and protein molecules into larger units partly depends, without doubt,
on the preparative procedures used in electron microscopy (de Robertis, 1956; Ryter
& Kellenberger, 1958; Davies & Spencer, 1962; Schreil, 1964). Difficulties in interpreting the structures seen in electron micrographs of thin sections are also due to the
inability to distinguish between molecules of DNA and protein and to the thinness of
the section compared with the size of the chromosome.
When chromosomes from a variety of species were disrupted on an air-water interface (Kleinschmidt, Lang, Jacherts & Zahn, 1962), Ris & Chandler (1963) and Wolfe
(1965) found long threads of diameter about 200-250 A, apparently without substructure. Hotta & Bassel (1965) claim to have seen circular DNA molecules from
disrupted nuclei of boar sperm and wheat-germ nuclei.
Our results, presented in this paper, relate to the surface structure of chromosomes
in thin sections of certain undisrupted cells from newt spleen. Although the interior
structure seen in the electron micrographs gives no evidence of definite organization
there are two types of surface structure, one of which, the sheets, has a remarkably
constant thickness. We believe that these surface structures provide important clues
about the nature of the constructional units used in the organization of the chromosome. A brief account has appeared elsewhere (Davies & Tooze, 1964).
The spleen of urodeles is the major site of erythropoiesis (Jordan & Spiedel, 1924,
1930; Jordan 1933), and this paper is one of several that deal with the distribution of
DNA (as estimated with Feulgen stain) in the mitotic chromosomes of erythroblasts, the changes in fine structure of the cells undergoing erythropoiesis, and
the fine structure of the non-erythrocytic cells of the spleen in the newt Triturus
cristatus.
MATERIALS AND METHODS
Small pieces of spleen from the newt T. cristatus were fixed in : (a) 1 % osmium
tetroxide in veronal-acetate buffer (pH 7-2), containing 0-14 M sucrose (Palade, 1952;
Caulfield, 1957) for 1 h; (b) 1 % osmium tetroxide in veronal-acetate buffer (pH 6-3),
containing 0-24 M sucrose and o-oi M calcium chloride, for 1-5-2 h (Davies & Spencer,
1962); (c) 1-5-5% ghitaraldehyde in o-i M phosphate buffer (pH 7-2) for 1-2 h,
Structure of mitotic chromosomes
333
washed overnight in o-i M phosphate buffer containing 0-2 M sucrose (Sabatini,
Bensch & Barrnett, 1963); or (d) as in (c) and further treated in 1 % osmium tetroxide
in o-i M phosphate buffer containing 0-2 M sucrose (Sabatini et al., 1963). Some blocks
were embedded in Epon (Luft, 1961) or methacrylate, but most were in Araldite
(Glauert & Glauert, 1958; Luft, 1961).
Sections were cut with glass or diamond knives on a Cambridge ultramicrotome and
mounted either on collodion-covered grids, a small amount of carbon then being
deposited on the surface of the section after staining, or directly on to grids with
collodion and carbon. Sections were stained with either a 2% aqueous solution of
uranyl acetate for 4 h, or with lead citrate for 8 min (Reynolds, 1963), or both these
solutions.
Sections were examined in a Zeiss EM 9, or a Siemens Elmiskop I with a 50-/*
objective aperture at magnifications of up to 80000 at 80 kV. Thick sections (0-5-1 /*),
serial to thin, were examined with the light microscope (C. Zeiss) either by phasecontrast or after Feulgen staining; this helped recognition of stages in mitosis. Thin
serial sections were studied in the electron microscope to obtain a three-dimensional
picture of the structures. The haemoglobin content of cells was measured with a
Joyce-Loebl microdensitometer on photomicrographs taken in violet light (A: 4047 A).
OBSERVATIONS
Cell types
In thin sections of newt spleen we find dividing cells in which the chromosomes
appear relatively light in positive phase contrast (Figs. 7, 9, 10). This appearance is
due to the high concentration of haemoglobin surrounding the chromosomes, about
60-80% of that found in the cytoplasm of mature erythrocytes, according to the
measurements in violet light. These cells are presumably the polychromatophil
erythroblasts. In other cells the chromosomes appear dark in positive phase contrast
(Fig. 6) and these cells must contain relatively little or no haemoglobin; no measurements in violet light were made. These latter cells are either basophilic erythroblasts,
that is erythrocytes at a still earlier stage of development, when the haemoglobin
concentration is low, or possible haemocytoblasts, that is, primitive cells or stem cells.
These different cell types and their interrelationship will be described elsewhere.
In any one spleen, dividing cells of one type predominate, that is they contain either
much haemoglobin, or relatively little or none, suggesting some degree of synchrony
in red blood-cell formation. There was considerable variation in the mitotic index
among animals killed at the same time, some spleens consisting almost entirely of
mature erythrocytes. Jordan & Spiedel (1930) also noted variations in the relative
numbers of red and white (lymphoid) cells in the spleens of Triturus viridescens and
attributed them to a seasonal fluctuation.
The polychromatophil erythroblast in interphase
Since most of our data on the surface structure of chromosomes were obtained from
dividing polychromatophils, the appearance of these cells during interphase will be
334
H- G. Davies and J. Tooze
described briefly. Characteristically the cytoplasm contains a high concentration of
haemoglobin (Tooze & Davies, unpublished observations). In the nucleus the DNA
is located mainly in large heterochromatic or condensed regions (Fig. 12). Typically,
large nucleoli are present and are divided into fibrillar and granular regions, both
regions containing small areas of DNA; this structure is similar to that commonly
found in other animal and plant cells. The nuclear sap (or interchromatin regions)
consists of numerous large granules ranging in diameter from about 400 A downwards,
embedded in an ill-defined matrix containing some fibrils. Some of these granules are
close to the chromatin but separated from it by a zone of lower staining, and might
correspond to the perichromatin granules described by Watson (1962). The cytoplasm
contains many ribosomes, mitochondria and a Golgi zone; we have not seen centrioles
in interphase polychromatophils. Endoplasmic reticulum is scarce. The nuclear
envelope appears as a white zone surrounding the nucleus, the space between the two
membranes often being extended in places, due no doubt to poor fixation and embedding. The membranes comprising the nuclear envelope are often difficult to distinguish
due to the high density of the adjacent haemoglobin and chromatin. The surface of the
interphase chromosomes varies, being occasionally smooth but frequently irregular
(Fig. 12) and bearing short projections of chromatin, whilst small blobs of chromatin
occur adjacent to the main mass of material. This irregular appearance resembles that
found in mitotic chromosomes.
The stages in the division cycle
Although it is easy to recognize the mitotic stages in squash preparations in the light
microscope, this is not so when examining thin sections of mitotic cells in the electron
microscope. Success in recognizing the stage in the division cycle depends on how many
chromosomes are contained in the section, whether the same cell can be found in
adjacent thick sections, and also upon the following factors. The interphase nucleus
of newt erythroblasts contains much condensed DNA; hence it is not possible to
recognize very early prophases as can be done in some other cell types, where the
interphase chromosomes are in an extended or uncondensed state (Ris & Mirsky,
1949) and therefore very different in appearance from the mitotic chromosomes. The
earliest recognizable stages of prophase are characterized in the electron microscope by
a diminution in the amount of chromatin on the interior surface of the nuclear envelope leaving either chromatin-free segments of envelope (Fig. 15), or, at the earlier
stage (Fig. 14), thinner zones of chromatin attached to it. In the Feulgen-stained
section through the prophase cell (Fig. 5) the density of the chromosomes is similar
to that of the interphase chromosomes (a in Fig. 8), but at this stage the mitotic
chromosomes are more uniform in diameter and the interchromosomal spaces more
extensive than in the interphase nucleus. At the beginning of prometaphase the
nuclear envelope is disrupted (Fig. 13), and at a later stage the chromosomes are more
widely spread, are randomly arranged throughout the cell, and most of them bear
attached fragments of nuclear envelope (Fig. 17).
A characteristic feature which allows cells to be classified as prometaphase or metaphase is the presence of pairs of chromatids clearly separated from one another.
Structure of mitotic chromosomes
335
When sectioned transversely they appear as figures-of-eight in the light microscope
(Figs. 6, 7) and frequently appear fused together in the electron microscope (Figs. 18,
22). All cells with such paired chromatids have been classed as prometaphase unless,
owing to fortunate sectioning, the chromosomes appeared regularly arranged with
respect to the spindle, when they were classed as metaphase; consequently some cells
classed as prometaphase may have been unrecognized metaphases. Such well-separated
pairs of chromatids were not seen at anaphase, which enabled the stages to be distinguished from one another. It was concluded that at anaphase the spindle is
sometimes bent, since in some cells the two daughter groups of chromosomes were
sectioned approximately longitudinally and transversely respectively (Fig. 9), while
in others both daughter groups were sectioned approximately transversely (b in Fig. 8).
Frequently the section contained only one group of daughter chromosomes, sectioned
transversely (Fig. 26).
In cultured cells, in mouse fibroblasts (Fell & Hughes, 1949) and in newt fibroblasts (Boss, 1959), where the sequence of events in an individual mitotic cell can be
followed in the light microscope, the anaphase chromosomes are seen to clump together,
parts of individual chromosomes sometimes projecting from the clump. After maximum clumping, signifying the end of anaphase, the individual chromosomes separate
from one another, when the spaces reappearing between them are seen to be bridged
by fine connecting filaments. At this stage of telophase the nuclear envelope is first
visible in the light microscope. At a later stage in telophase, vacuoles or internal spaces
appear within the chromosomes. The latter gradually lose their individual identity
during nuclear reconstruction. These observations on living cells have enabled us to
identify similar stages in thin sections of polychromatophil erythroblasts. At early
telophase (Fig. io), due to sectioning, parts of individual chromosomes may appear
disconnected from the main mass of clumped chromosomes. The chromosomes in the
electron micrograph (Fig. 40) of a cell at a similar stage contain numerous small spaces
within the chromatin; small segments of nuclear envelope are attached to the chromosome surface. Later, the individual chromosomes reappear, and the spaces between
them are occasionally crossed by Feulgen-positive filaments (Figs. 11, 42). The spaces
within and between chromosomes enlarge considerably (Figs. 41, 42). In the telophase
cell shown in Fig. 41, the nucleus is completely enveloped by membrane. During the
reconstruction of the daughter nuclei in the polychromatophil erythroblasts of the
newt, the chromosomes must, at least partially, retain their individuality, thus producing the condensed DNA of the interphase nucleus and the frequent irregularity of
the nuclear outline.
Surface structure of chromosomes in newt A
In the spleen of newt A, from which most of the numerical data have been obtained,
mitotic divisions were frequent and restricted almost entirely to one type of cell,
the polychromatophil erythroblast. One half of this tissue was processed in glutaraldehyde only and the rest post-fixed in osmium tetroxide prior to embedding; this
permitted direct comparison of the results of the two procedures. The irregularities in
outline of the chromosomes in these cells are due either to parts of the chromosomal
22
Cell Sci. 1
336
H. G. Dairies and J. Tooze
substance projecting from the surface, or to projections of chromosomal material
which are limited by membranes.
Projections. At prometaphase (Figs. 17-19 and 21), and metaphase (Fig. 22) the
irregularities on the surfaces of the chromosomes are rather well defined. They appear
as finger-like processes or projections (Fig. 18). Some processes have a constant width,
the value varying somewhat in different projections. Others take the form of a fine
thread rising out of the chromosome, ending in a blob equal in diameter to the width
of the widest projections. Rarely, the projections are Y-shaped (Fig. 22). The disconnected blobs adjacent to chromosomes, which are particularly distinct in the cells
at prometaphase and metaphase (Figs. 18, 19, 22), are presumably sections through the
finger-like processes and are most numerous when the surface of the chromosome is
sectioned (Fig. 19). The processes are frequently circular or roughly rectangular in
cross-section. These observations are consistent with the view that many of these projections are indeed finger-like; that is, roughly cylindrical in shape, the axis of the
cylinder not necessarily being straight. The variation in appearance may be attributed
to sectioning.
The chromosomes at anaphase also have an irregular surface (Figs. 25, 35)
due to numerous projections. At the end of anaphase or beginning of telophase,
when the chromosomes are maximally clumped (Fig. 40), their surface is relatively
smooth.
The prophase chromosomes of Figs. 14 and 15 have relatively smooth surfaces, but
in many other cells at prophase the outline of the chromosomes is very irregular.
At the beginning of prometaphase (Fig. 13), when the nuclear envelope is breaking into
fragments, the surfaces of the chromosomes are irregular, numerous projections being
present. We have not yet studied prophase chromosomes in sufficient numbers to be
sure whether the variation in surface regularity is real or depends on the method of
fixation. In mitotic cells at prometaphase, metaphase, and anaphase, however, a small
proportion of those fixed in glutaraldehyde alone contained relatively smooth chromosomes, with only few, but nevertheless well-defined, finger-like projections. This suggests different methods of fixation and embedding may vary in their effect on the
surface structure of chromosomes.
A rough estimate of the diameters of the projections in cells at prometaphase and
metaphase was made as follows. The diameters of all the unattached circular blobs
easily recognizable as chromatin, the minor diameters of the unattached rectangular
blobs, and the widths of the projections attached to chromosomes, were measured on a
single micrograph. Obviously some of these values are too low because only part of a
projection was contained in the section. In a cell at prometaphase (Fig. 19 and similar
micrographs), fixed in both glutaraldehyde and osmium tetroxide, most of the diameters
ranged from about 700 to 1400 A, with an average value ofiiO5±2ioA (Fig. 1C). In
two other cells at prometaphase and metaphase fixed in glutaraldehyde alone, the
individual average values were similar and the diameters ranged from about 600 to
1200 A (Fig. 1D), with an average for both sets of data of 925 ± 185 A; that is, about
16% lower than the previous average. The lengths of the longest projections encountered were equal to about half the diameter of the chromosome.
Structure of mitotic chromosomes
337
Newt A also contained a few mitotic cells in which the chromosomes appeared dark
in phase contrast; that is, the cells contained relatively little or no haemoglobin. In
the anaphase cell shown in Fig. 29 the surfaces of the chromosomes bear projections
similar to those found in dividing polychromatophils. That this cell contains little
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Fig. I. The diameter of the chromosomal projections, as a function of the number of
projections. A, in an anaphase cell from newt B containing little or no haemoglobin
fixed in i % OsO4 + o-oi M Ca1+; measurements on serial sections average 890 ±
200 A. B, in a polychromatophil from newt D at metaphase; measurements on serial
sections average 1115 ± 160 A. C, in a polychromatophil from newt A at prometaphase;
measurements on a single section average 1105 ± 210 A. D, in polychromatophils from
newt A at prometaphase and metaphasefixedin glutaraldehyde only, combined average
925 ± 185 A.
or no haemoglobin is indicated by the low visibility of the region between the two
membranes of the endoplasmic reticulum or fragments of nuclear envelope, the
membranes themselves appearing more clearly. Apparently, in the polychromatophils,
the haemoglobin has the same effect as a negative stain.
Membrane-limited projections (sheets). These structures commonly take the form of a
long projection of chromosomal material of constant width, bounded on either side by
338
H. G. Davies andj. Tooze
a white zone resembling the nuclear envelope (Fig. 28). The visibility of the two
membranes forming the sides of each white zone is often low, as indeed is that of the
membranes comprising the nuclear envelope, for reasons already discussed.
From the continuous nature of the projections in most micrographs it seems
reasonable to suppose that they are sections through projecting sheets, rather than
cylinders. This was established by serial sectioning. For example, in Figs. 30—35 the
projection is seen in a series of 6 sections of a cell at anaphase. Since these were silvercoloured sections, about 600 A thick, the sheet extends in depth to at least 0-36 /i,
comparable to the radius of the chromosome itself. Since the projections are often
several microns long the sheets must be quite extensive structures. We assume that
the zones limiting the sheet are fragments of nuclear envelope which remain attached
to the chromosome, and therefore refer to these structures as envelope-limited or
membrane-limited sheets of chromatin. The small amount of endoplasmic reticulum
in the interphase polychromatophil is consistent with the view that these attached
fragments are part of the nuclear envelope. It is reasonable to suppose that the sheet
bounded by the light zones is chromosomal material or chromatin, since it has the
same staining properties as the main body of the chromosome, a similar fine structure,
and appears to be continuous with it.
The membrane-limited sheets have been found in newt A at prometaphase (Figs. 18,
21, 45), metaphase (Fig. 22), anaphase (Figs. 25, 27, 28, 35, 43, 46-48) and telophase
(Figs. 40, 44); that is at all stages of division except prophase. Of the latter stage,
however, we have only a few micrographs.
There are variations in the geometrical arrangement of the sheets with respect to the
chromosome, and in the membranes limiting the sheets. From a geometrical viewpoint
there are two extreme ways of arranging a sheet, depicted simply as a ribbon, with
respect to a cylinder (Fig. 2 A). The axis of the cylinder may be parallel to the plane
of the sheet, the angle d between the line xx x2 and the tangent plane to the cylinder
(Fig. 2 A, a) being variable. Alternatively the arrangement can be shown at b, the angle
<f> between y^y^ and the axis of the cylinder being variable. In a transverse section
through the cylinder, configuration a would appear as indicated in Fig. 2 B; the probability of seeing the sheet in configuration b in such a transverse section would be
small. Conversely, in a longitudinal section through the cylinder, configuration b
would appear as indicated in Fig. 2 C, and the probability of seeing configuration a in
such a section would be small. Although it is not always easy to decide the direction
of the main chromosome axis, no preferential orientation seemed to exist. The metaphase chromosome in Fig. 22 bearing a sheet corresponds to Fig. 2 B, the sheet being
at an angle of about 900; the anaphase chromosome in Fig. 35 corresponds to Fig. 2C,
the angle between sheet and chromosome being about 200.
A single chromosome may carry two sheets (Figs. 25, 27), and rarely two sheets may
exist side by side as in the anaphase chromosome of Fig. 46, both sheets apparently disconnected from the main part of the chromosome. In some non-serial sections through
the same anaphase chromosome the sheet appears either connected at one end to the
chromosome (Fig. 25 (s3), Fig. 47), or adjacent but unconnected to the chromosome
(Fig. 48). Breaks in the continuity occur infrequently (Figs. 40, 51). Not uncommonly,
Structure of mitotic chromosomes
339
the sheet may appear connected to the chromosome, not at its end but at some distance along it, as in the anaphase chromosome in Fig. 43.
The envelopes limiting each side of the sheet usually extend considerably beyond it,
sometimes running approximately parallel to one another (Fig. 48) but frequently
diverging (Fig. 43). In the complex arrangement of membranes on the anaphase
chromosome of Fig. 28, the envelope limiting one sheet also limits another.
Fig. 2. A, Schematic diagram showing a cylinder (the chromosome) to which are
attached sheets, simply shown as ribbons, arranged in two possible but extreme
orientations, a and b. B, a transverse section through the cylinder at a. C, a longitudinal section through the cylinder at b.
In the interphase polychromatophil erythroblasts and in nearly mature erythrocytes,
envelope-limited sheets occur on the surface of the nucleus (Figs. 49, 51-53), proecting into its interior (Figs. 50, 53), or protruding from the surface (Figs. 53, 54).
Although the dimensions in the plane of the chromatin sheet are variable, it is
striking that the thickness or width of the chromatin sheets is approximately constant
at all stages of division, as well as in interphase cells. After fixation in glutaraldehyde
and osmium tetroxide (Fig. 3 A-E) the average width is 465 A. After fixation in glutaraldehyde only (Fig. 3F), the average thickness is 315 ± 30 A, that is, about 32% less.
After fixation in glutaraldehyde only, the space between the two membranes comprising the envelope limiting either side of the sheets was frequently more extended than
when post-fixation in osmium tetroxide was employed. Blocks of newt spleen fixed in
glutaraldehyde alone were also more difficult to section.
In the few dividing cells found in this spleen, which contained relatively little or no
haemoglobin, sheets of chromatin were not seen.
H. G. Davies and J. Tooze
34°
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Fig. 3. The width of the chromatin in the envelope-limited sheets as a function of
number of sheets. Each value is the mean of 3 measurements. A, interphase, average
465 ± 40 A. B, prometaphase, average 475 ± 20 A. C, metaphase, average 465 ± 30 A.
D, anaphase, average 470 ± 30 A. E, telophase, average 450 ± 25 A. Cells in A to E fixed
in glutaraldehyde (5 %) + OsO4; cells in F, glutaraldehyde (5 %) only. F, all stages of
mitosis, average 315 + 30 A. In the histograms A to E the unnumbered rectangles refer
to measurements on a sheet seen once, whilst the similarly numbered rectangles refer to
a particular sheet seen in a series of sections through the same chromosome. For an
example, the two numbers 7 in histogram D refer to measurements on two sheets;
there is no relationship between these numbers and the numbers used to refer to sheets
in the plates. In the histogram F the letters within the rectangles refer to stage in
division; I, interphase; PM, prometaphase; M, metaphase; A, anaphase; the numbers following the letters have the same significance as in histograms A-E.
Surface structure of chromosomes in other newts
In the spleen of newt A the mitotic index was relatively high and sheets were common. Thus a section might contain as many as 10 mitotic figures and, in up to half
this number, one or more sheets per cell were seen.
Newt B. In the spleen of another newt, B, with mitotic index similar to that of
newt A, fixed in osmium tetroxide + o-oi M calcium chloride the dividing cells contained little or no haemoglobin. No sheets were encountered despite extensive study,
but finger-like projections were seen similar to those in newt A. From one block of
tissue 6 serial sections were cut. In one micrograph a projection was selected and micrographs on either side were examined to see if the whole of the projection was contained
in the first selected micrograph. In this way, diameters were measured and found to
range from about 400 to 1400 A, with an average of 890 ± 200 A (Fig. 1 A). This wide
spread of values, apart from experimental error, might be associated with the relatively
poor quality of this fixation and some difficulty in sectioning. A few projections, however, had a diameter significantly lower, about half the average value. Disconnected
blobs could be traced back to the main part of the chromosome and in several instances
Structure of mitotic chromosomes
341
the appearance of a thin thread connecting a thicker blob to the chromosome was seen
to arise from sectioning a relatively uniform cylindrical projection. Difficulties in
building up a good three-dimensional picture of the surface of the chromosomes was
attributed to small distortions caused by sectioning, which were not appreciated until
the attempt at reconstruction was made. Distortion in the microscope lenses was
greatly reduced by proper choice of operating conditions. These observations, however,
showed clearly that the projections did not return to the main part of the chromosome, forming loops as in the lampbrush chromosomes (see Callan & Lloyd, i960).
The absence of loops in single micrographs is consistent with this conclusion.
Newt C. The spleen was fixed in 1 % osmium tetroxide, and embedded in methacrylate. In the dividing cells the concentration of haemoglobin was high, similar to
that found in newt A, resulting in relatively light chromosomes in electron micrographs stained with lead citrate only. In a few instances—for example, the anaphase
cell in Fig. 38—the outline of the chromosomes was found to be irregular, owing to
projections apparently similar to those encountered in other newt spleens fixed
according to different procedures.
Newt D. Newt D had a high mitotic index restricted mainly to polychromatophil
erythroblasts and after an extensive search only two membrane-limited sheets of
chromatin were found, although finger-like projections were present. The large number of these projections caused transverse sections through anaphase chromosomes
(Fig. 26) to present a somewhat bizarre appearance. In 3 serial sections of a cell at
metaphase fixed in glutaraldehyde plus osmium tetroxide, measurements of projection diameter were made, thereby avoiding uncertainty due to sectioning part of a
projection. The diameters ranged from about 800 to 1300 A, with an average value of
1115 ± 160 A (Fig. iB), which is similar to that obtained on newt A (Fig. iC). We
encountered some variability in the appearance of chromosomes through the blocks
of tissue. In some cells the chromosome material seemed less closely packed and
sections through the projections less well defined (Fig. 37).
Newt E. Newt E had a high mitotic index restricted mainly to polychromatophil
erythroblasts. No sheets were found but only a few observations were made. The
chromosomal projections were similar to those of other newts.
Newt F. In the spleen of this newt divisions were found among polychromatophil
erythroblasts. A few observations on the mitotic cells indicated that envelope-limited
sheets were common and the dimensions similar to those reported for newt A.
Although spleens from many other newts were examined, dividing cells were uncommon. In another amphibian, Rana esculenta, a chromatin sheet was found attached
to the nucleus of an interphase neutrophil (Fig. 39).
The attachment of nuclear-envelope fragments to chromosomes
Long fragments of nuclear envelope-like material remain attached to the chromosomes in newt A during prometaphase (Figs. 17,18), metaphase (Fig. 22) and anaphase
(Fig. 35). Frequently these fragments are situated at the periphery of the cell. No doubt
some of the material scattered through the dividing cell is endoplasmic reticulum,
although, as already noted, endoplasmic reticulum is scarce in the cytoplasm of the
342
H. G. Davies and J. Tooze
polychromatophil erythroblast at interphase. In the spleen of newt D, in which
dividing polychromatophils are common but envelope-limited sheets rare, fragments
of envelope-like material also remain attached to the chromosomes during division, so
that there does not appear to be any simple correlation between persistence of attachment of nuclear envelope and the presence of sheets. Other factors may be involved,
however. If the persistent attachment of envelope fragments to the mitotic chromosomes were a necessary, though not sufficient factor, for the formation of envelopelimited sheets, then this might account for the singular absence of sheets on chromosomes from most other species where, in fact, persistent attachment of fragments of
nuclear envelope has not been noted. In dividing cells containing relatively little or no
haemoglobin (newt B) attachment of nuclear envelope to chromosomes was infrequent
but it has been seen, for example, in such a cell at anaphase from newt A (Fig. 29).
Other observations on dividing cells
Spindle. In dividing polychromatophils, after staining in uranyl acetate plus lead
citrate, the spindle appears as a region of lower density connecting the chromosomes
with the poles (Fig. 22). Individual spindle tubules can only just be made out at higher
power, the low contrast being due to the haemoglobin, but, where spindle elements
enter the substance of the chromosomes, less intensely stained tubular regions, about
250 A in diameter, are clearly seen (Fig. 27). Robbins & Gonatas (1964) have described the passage of spindle tubules through metaphase chromosomes in Hela cells.
Centrioles and kinetochores. During our studies only two centrioles were seen in
dividing polychromatophil erythroblasts. In the anaphase cell fixed in osmium
tetroxide and stained with lead citrate, the centriole is surrounded by a more densely
staining zone similar to that described in Hela cells by Robbins & Gonatas (1964) and
the kinetochore is also stained (Fig. 38). In a polychromatophil (not illustrated) at
anaphase fixed in glutaraldehyde plus osmium tetroxide and stained with uranyl
acetate plus lead citrate, no densely staining zone, but a zone free from ribosomes,
surrounded the centriole. The centrioles from interphase basophilic erythroblasts in
the newt spleen have a similar ribosome-free zone (Tooze & Davies, unpublished
observations). Although anaphase chromosomal configurations similar to the one in
Fig. 38 were frequently encountered in polychromatophils fixed in glutaraldehyde
plus osmium tetroxide and double-stained, kinetochores were not seen. These few
observations suggest that visualization of these structures might depend on fixation
and staining procedures.
Chromosomefinestructure. After fixation in glutaraldehyde plus osmium tetroxide
and staining in uranyl acetate plus lead citrate, the interior of the chromosome appears
as an irregular network which contains, as its finest component threads and granules
(which may be cross-sections through threads), about 20-40 A in diameter (Fig. 36);
the threads often pursue a zig-zag course. These finest components sometimes appear
to be aggregated into larger units, varying in size up to several hundred angstrom
units. There is no sign of a regular arrangement.
The nuclear granules. In the interphase and prophase nuclei of polychromatophils
(Figs. 12, 14, 15), there is a striking abundance of large granules in the interchromatin
Structure of mitotic chromosomes
343
spaces. The large granules occur at other stages of erythropoiesis and in leucocytes
from spleen (Tooze & Davies, unpublished observations). They range in diameter
from about 400 A downwards, part of the size variation being no doubt due to sectioning, since the dimensions of the section are comparable with those of the largest
granules. After double staining the interchromatin granules appear less intensely
stained (Fig. 16) than the cytoplasmic ribosomes, which are about 200 A in diameter.
The ribosomes also have a somewhat variable contrast, presumably due to sectioning.
A few observations have been made on the behaviour of the large granules during
cell division. At prophase, before breakdown of the nuclear envelope, a clear distinction is seen between the large interchromatin nuclear granules and the cytoplasmic
ribosomes (Fig. 16). After fragmentation of the envelope, the two types of particle are
scattered through interchromosomal space, and groups of large granules, reminiscent
of polysomes, can clearly be distinguished (Figs. 23, 24). At the end of anaphase or
beginning of telophase the chromosomes are maximally clumped and contain only
small spaces (Fig. 40), devoid of granules. As telophase proceeds these spaces enlarge
and are seen to contain large granules (Fig. 41); in this cell large granules could not be
detected in the cytoplasm.
DISCUSSION
Our first finding is the membrane-limited sheets of chromatin attached to the
mitotic chromosomes in dividing polychromatophil erythroblasts in the spleen of certain
newts only. Sheets also occur in the interphase nuclei of these animals. We wish to
stress their constant width, though the exact dimension depends on the nature of the
fixative; thus after fixation in glutaraldehyde plus osmium tetroxide, the width of the
chromatin was 465 A; after glutaraldehyde only, 315 A. There are several reports of
membrane-limited projections attached to chromatin in other species, and it is likely
that these are basically similar to the structures we describe in this paper. The widths
of the chromatin projections have been measured in the published micrographs. In
human neutrophils, in a case of D1(i3-i5)-trisomy syndrome, Huehns, Lutzner &
Hecht (1964) figure a membrane-limited projection extending from the interphase
nucleus, the chromatin being about 480 A thick. Dr Huehns kindly showed us micrographs taken by Dr Lutzner in which the average of 5 projections was about 520 A.
These characteristic projections have not been seen in normal human neutrophils
(Huehns, private communication). Epstein & Achong (1965) and Epstein, Barr &
Achong (1965), in a study of human lymphoblasts in two tissue culture-strains
(EB 1, EB 2) of Burkitt's lymphoma, found spectacular membrane-limited extensions
of the chromatin of the interphase nucleus about 300 A thick, after fixation in glutaraldehyde plus osmium tetroxide. In a telophase nucleus from a flagellated plant cell,
Prymnesium parvum (Manton, 1964), a piece of chromatin about 380 A thick is shown
limited on both sides by the nuclear envelope. Thefixationmedium was glutaraldehyde
followed by osmium tetroxide. Although the data are limited, the thickness of the
sheets appears to range from about 300 to 500 A. Part of this variation may be due to
the sensitivity of the structures to the chemical environment, which we have described.
344
H. G. Davies and J. Tooze
In the human lymphoma, however, the width appears to be significantly lower than
in human neutrophils or in cells from other species, after fixation in a similar medium.
The constant thickness of chromatin in the sheets from one species, the similarity in
their morphology from species to species, and the approximate similarity in their
thickness suggest that the structures have some general significance.
Our second finding concerns the irregularity in outline of the chromosomes, which
takes the form of finger-like projections visible from prometaphase to anaphase. This
irregularity is a constant feature of chromosomal morphology in dividing cells from
spleens of all newts; it occurs in cells containing much haemoglobin—that is, polychromatophil erythroblasts—and in dividing cells containing relatively little or no
haemoglobin. Other workers have found irregularities in the surface structure of
chromosomes examined in the electron microscope. Projections similar to those reported
here have been described by Barnicott & Huxley (1965) in newt fibroblasts grown in
tissue culture. Less-pronounced surface irregularities occur in chromosomes from onion
root-tip (Porter & Machado, i960), bean root-tip (Lafontaine & Chouinard, 1963)
and in cultured cells of salamanders (Bloom & Leider, 1962). The dimensions of the
finger-like projections on chromosomes from newt spleen are below the limits of resolution of the light microscope. They appear to be different from the filamentous bridges
between chromosomes described in the light-microscope studies of Boss (1959) and
Fell & Hughes (1949) already referred to. Projections on the surface of early prophase
chromosomes in primary spermatocytes of the salamander are illustrated by Wilson
(1925, page 123) but they are not shown at prometaphase.
Although the data obtained so far do not permit conclusive answers to questions
concerning the origin and significance of the envelope-limited chromatin sheets,
certain suggestions can be made.
First, concerning the origin of the sheets, we suppose that the limitation by nuclear
envelope of both sides of the sheet is most likely to occur when the envelope has a
dynamic relationship with the chromatin; that is, either at prophase during the disruption of the nuclear envelope, or at telophase during its reformation. The fate of the
nuclear envelope during cell division, as seen in the electron microscope, has been
discussed by Moses (1958), Porter & Machado (i960), Barer, Joseph & Meek (i960),
Jones (i960), and Robbins & Gonatas (1964). In late prophase it breaks up into
fragments and in cells of onion root-tip the endoplasmic reticulum-like fragments
occupy a zone surrounding the spindle, subsequently passing to the poles at metaphase
(Porter & Machado, i960). Fragmentation of the nuclear envelope and passage of
fragments to the poles was not observed in Hela cells by Robbins & Gonatas (1964).
They found during prophase an atypical projection of the nuclear envelope, perhaps
containing chromatin, directed towards the centriole, which resembles the structures
we have described. Robbins & Gonatas (1964) also report wave-like irregularities in
the nuclear envelope. In rat proerythroblasts and basophil erythroblasts, Jones (i960)
concluded that the nuclear envelope does not disintegrate and vanish rapidly during
cell division but persists although not attached to chromosomes. The fragments of
nuclear envelope are more complex and thicker than the endoplasmic reticulum seen
in the interphase cells. Porter and collaborators (1965, unpublished observations)
Structure of mitotic chromosomes
345
have recently elucidated the structure of the fragments of nuclear envelope in rat
erythroblasts and shown them to be triple-layered, that is presumably formed by a
juxtaposition of two pieces of envelope, the adjacent membranes fusing together to
form a relatively denser line. We do not find such triple-layered fragments in our
material. The structures observed by Jones (i960) and Porter et ah (1965, private communication) might be regarded as special forms of our membrane-limited chromatin
sheets in which the chromatin is absent.
Returning to the origin of the sheets of chromatin and their limitation on both sides
by the nuclear envelope, we suggest that this occurs during late prophase or early
prometaphase. We envisage that the large pieces of heterochromatin attached to the
nuclear envelope during interphase become reorganized during prophase into the
mitotic chromosomes, but that a layer or sheet of material, about 450 A in thickness,
remains attached to a segment of the interior membrane of the envelope. To the membrane-free surface of this chromatin sheet another piece of nuclear envelope becomes
attached. This second layer might be either a folded-back part of the original fragment
or a separate fragment of nuclear envelope, the movement of the chromosomes during
mitosis favouring the transposition of membranous material. More complicated interfoldings of envelope and chromatin sheet would be required to form the doublelayered structures seen in some cells (Fig. 46). Some support is given to this idea by
the existence of a layer of chromatin of about the correct thickness on the interior
surface of the cell in prophase (Fig. 14). When the chromatin is sandwiched between
the two envelopes it is presumably stabilized so as to resist the forces normally operating
in chromosome assembly. According to this idea, the membrane-limited sheets have no
special function, but could provide clues to the organization of chromosomes. The
sheets so formed would persist throughout division and the following interphase.
Epstein & Achong (1965) have suggested that the envelope-limited sheets found in
interphase cells from a culture of Burkitt's lymphoma may arise as a result of an
abnormality of cell division. We assume that this may be at telophase during the
application of the new nuclear envelope, since no sheets are found on the mitotic
chromosomes at earlier stages (Epstein, private communication). Another possibility
is that the nuclear extensions are formed during interphase in this cell type; membrane
limitation of sheets may not occur at the same stage in all species.
The fact that, in the newt, the thickness of the sheets of chromatin is constant after
a particular preparation procedure probably signifies some constant constructional
unit in the protein and DNA of the chromatin. It is unlikely that the thickness of the
sheet of chromatin is entirely a property of the nuclear envelope by which it is limited
but it is, of course, possible that the adjacent membranes modify or partly determine
the orientation and dimension of the hypothetical constructional unit just mentioned.
One hypothesis which could account for the dimensions of the chromatin sheet as
well as for those of the majority of the projections is that the chromosomes contain a
thread about 450 A diameter; this figure is based on the values obtained after fixation
with glutaraldehyde plus osmium tetroxide. This hypothetical thread might consist
either of a bundle of DNA molecules and protein (Steffenson, 1959; Kaufmann et ah,
i960) or of only one or two DNA molecules coiled several times in association with
346
H. G. Davies and J. Tooze
protein (see Swift, 1962a). On further coiling of this 450-A thread an irregularity is
assumed to occur (Fig. 4A) whereby a portion of the thread loops outwards, the individual elements twisting on themselves to produce a projection of diameter somewhat
greater than twice that of the thread; projections of average diameter 1100 A were
actually found. The spread in the values of the diameters might be due partly to variations in the degree of coiling. Further coiling would be needed to form the chromosome.
To give an example, when a thread of DNA and protein, say 30 A in initial diameter,
is coiled three times, its diameter increases to 470 A, assuming an increase in diameter
of x 2-5 after each coiling. When this intermediate unit is again coiled 3 times it
Fig. 4. A, showing how a thread may be coiled up, part of the thread forming projections (arrows) of diameter somewhat greater than twice that of the thread. B, a thread
laid down on a surface (su) and close-packed forms a sheet (s).
attains a diameter of 0-7 /i, roughly comparable to that of the chromosome. The sheets
may be formed by an alignment of the threads (Fig. 4B) on the surface of the nuclear
envelope. Sheets could be formed at telophase when new nuclear envelope is applied
to the chromatin, supposedly partially uncoiled into a mass of threads of diameter
45° A.
We wish to emphasize that we do not have any experimental evidence for this hypothesis which seeks to relate two sets of numerical data on surface structure. These
suggestions, which assume the chromosome to be built up by multiple coiling, do not
explain why an irregularity of coiling occurs primarily at the stage of the 450-A
thread so that the majority of the projections have an average diameter somewhat
greater than twice this value. Furthermore, threads of this diameter—that is, 450 A—
are not seen inside intact chromosomes nor are they found after disruption on a
water surface, when the threads are about 200-250 A in diameter (Ris & Chandler,
1963; Wolfe, 1965). We have already noted the apparent fusion of metaphase chromatids, a demarcation zone being absent. This lack of a distinct boundary between
nucleoprotein units may be responsible for the difficulty in distinguishing individual
threads inside chromosomes. Another factor that would diminish visibility would be
the presence within the chromosomes of substances from the surrounding medium.
Granules between the chromatin regions in the nuclei of various interphase cells
have been described by numerous workers, in amphibia in particular by Fawcett
(1955) and Swift (1963). The granules of the nucleus have recently been discussed by
Granboulin & Bernhard (1961), Bernhard & Granboulin (1963) and Swift (1963).
Structure of mitotic chromosomes
347
In the nuclei of ascites cells, Swift (1963) has described large ribonuclease-extractable
particles about 200-500 A in diameter, and small particles about 100 A in diameter
which were not ribonuclease-extractable. During cell division the small particles
persisted between the chromosomes and gradually disappeared from the cytoplasm
of the daughter nuclei as they reappeared inside the nucleus. Our observations on the
behaviour of the large granules in newt polychromatophil erythroblasts parallel those
of Swift (1963) on small particles. The fact that these large granules occur in groups
reminiscent of ribosomes is interesting in relation to the possible role of nuclear
particles in nuclear protein synthesis (Allfrey, 1963). Since the large granules scattered
throughout the cell in division do not remain in the cytoplasm of the daughter cells,
we must assume either that they are broken down in the cytoplasm and resynthesized
by the telophase nuclei or that they are reabsorbed by the telophase nuclei. A protein
fraction which is normally present within the interphase nucleus and is scattered
through the mitotic cell, reappearing exclusively in the nuclei of the daughter cells,
has been reported by Goldstein (1963) and Prescott (1963). Our observations and those
of Swift (1963) may be related to this phenomenon.
We are greatly indebted to Professor Sir John Randall, F.R.S. for his continued encouragement, as well as to Dame Honor B. Fell, F.R.S., Professor M. H. F. Wilkins, F.R.S., and
Dr B. M. Richards for discussions. We thank Miss P. Rush for unfailing technical help, and
Mrs F. Collier and Miss M. Blade for help in preparing the illustrations.
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ABBREVIATIONS
b
c
ca
ce
ch
d
db
er
f
g
h
blobs of chromatin
cytoplasm
isolated chromosome segment
centriole
chromatin, chromatid, or chromosome
discontinuity
dark body
endoplasmic reticulum
filament
granules within nucleus
space within chromosomes
k
m
n
ne
nu
P
Pi
r
s
sp
su
t
kinetochore
mitochondrion
nucleus
nuclear envelope
nucleolus
projections
polysomes
ribosomes
sheet of chromatin
spindle
surface
thread-like structure
All cells except Fig. 39 are from the spleen of the newt, and unless otherwise
stated were fixed in glutaraldehydc (5%) + OsO4, stained with uranyl acetate plus
lead citrate and embedded in Araldite.
Fig. 5. A Feulgen-stained i-fi section of a polychromatophil erythroblast at prophase ; bright-field, x 3 900.
Fig. 6. An unstained i-/i section of a metaphase cell at a developmental stage with little
or no haemoglobin. Pairs of chromatids appear as figures-of-eight (arrows) and the
spindle as a relatively dark region. OsO< + o-oi M Ca>+, phase-contrast, x 3300.
Fig. 7. An unstained 1 -/* section through a polychromatophil erythroblast at metaphase.
Pairs of chromatids (arrows) are light compared with those in Fig. 6 because of the
high concentration of haemoglobin in this cell. Phase contrast, x 2000.
Fig. 8. A Feulgen-stained i-fi section through polychromatophil erythroblasts at interphase (a) and at anaphase (b); the two groups of daughter chromosomes in b are
sectioned approximately transversely. Bright-field, x 3 900.
Fig. 9. An unstained i-/< section through a polychromatophil at anaphase, one group
of daughter chromosomes (long arrows) being sectioned approximately longitudinally,
the other transversely (short arrows). Phase contrast, x 3 900.
Fig. 10. An unstained i-fi section through a polychromatophil at the end of anaphase,
showing one group of daughter chromosomes clumped together with, however, a few
chromosome arms projecting outwards and appearing as isolated segments (ca) owing
to sectioning. A few small holes (arrow) appear within the main mass of chromatin.
Phase contrast, x 3 200.
Fig. 11. A Feulgen-stained i-/i section of a polychromatophil at a stage of telophase
when the individual chromosomes separate again, filaments (arrows) appearing to
connect them. Bright-field, X3900.
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(Facing /?. 350)
Fig. 12. A low-power electron micrograph showing some of the structures encountered
in the interphase polychromatophil erythroblast. The large dark masses inside the
nucleus are the interphase chromosomes, the surface of which is irregular and bears
a few projections; there are adjacent blobs of chromatin. Scattered throughout the
interchromatin regions are dense granules. Small fragments of nucleoli are embedded
in the chromatin. The nuclear envelope appears as a white zone. The black dots scattered throughout the cytoplasm are ribosomes. There are numerous mitochondria,
a dark body and very little endoplasmic reticulum. x nooo.
Fig. 13. Electron micrograph of a polychromatophil at the beginning of prometaphase
when the nuclear envelope is disrupted in places (arrows). The surfaces of the
chromosomes are irregular and there are numerous blobs of chromatin adjacent to,
but separated from, them. Glutaraldehyde (1-5 %) + OsO4. x 6800.
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Fig. 14. Electron micrograph of a polychromatophil at what is thought to be early
prophase, showing thin zones of chromatin (arrows) similar in appearance and dimensions to the envelope-limited sheets except that only one surface is bounded by envelope.
A nucleolus is still present and numerous granules occur between the chromosomes.
Spaces appear within the chromosomes, x 16000.
Fig. 15. Electron micrograph of a polychromatophil at prophase. A few spaces occur
within the chromosomes; large segments of the envelope are free of chromatin.
Glutaraldehyde (1-5 %) + OsO4. x 14800.
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Fig. 16. Electron micrograph of part of the prophase cell in Fig. 15, showing the
large granules (arrows) in the nucleus. The cytoplasm contains many ribosomes, the
closeness of packing making it difficult to distinguish polysomes. Fragments of chromatin are attached to the nuclear envelope, x 42000.
Fig. 17. Electron micrograph of a polychromatophil at prometaphase. Chromosomes
are scattered throughout the cell and long fragments of nuclear envelope (arrows)
remain attached to them. The surfaces of the chromosomes bear projections and there
are adjacent, apparently disconnected, chromatin blobs, x 15000.
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Fig. 18. Electron micrograph of a polychromatophil at prometaphase; pairs of chromatids appear fused as at chlt cli2l and c/;3, cli4. The projections (arrows) on the surfaces
of the chromosomes are numerous and well defined and there are adjacent blobs.
Note also the envelope-limited chromatin sheet and nuclear envelope, x 10600.
Fig. 19. Electron micrograph of part of the prometaphase seen in Fig. 18, showing,
at higher power, many blobs due to sectioning a chromosome surface; some are
circular in outline (&]), some approximately rectangular (b.>). The numerous minute
dots are ribosomes. x 21 000.
Fig. 20. An enlargement of the area circled in Fig. 19, showing that the polysome configuration of some of the ribosomes is maintained during cell division, x 62000.
Fig. 21. Electron micrograph of part of the prometaphase (Fig. 14) showing, at
higher power, the envelope-limited sheet and nuclear envelope-like fragment,
x 23000.
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Fig. 22. Electron micrograph of a polychromatophil at metaphase, showing the spindle,
a relatively light zone. Pairs of chromatids, chl, clu and ch3, c//4, appear fused together,
On the chromosome surfaces are projections, appearing occasionally Y-shaped (/>,),
or as a fine thread (p2), connecting a blob of chromatin. Blobs (arrows) are numerous.
Glutaraldehyde (5 %) only- x 10000.
Figs. 23, 24. Electron micrographs of part of a polychromatophil at metaphase showing
groups of large granules (arrows) and thread-like structures. The numerous small black
dots are ribosomes. Glutaraldehyde (5 %) only, x 40000.
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Fig. 25. Electron micrograph of a polychromatophil at anaphase. The chromosome
surface is irregular and there are envelope-limited sheets s,, .?., and s;1. x 8400.
Fig. 26. Electron micrograph of a polychromatophil at anaphase consisting mainly of
one group of daughter chromosomes sectioned transversely. Each chromosome bears
numerous projections. Note nuclear envelope-like fragments (arrows), x 5000.
Fig. 27. Electron micrograph of a section, serial to that in Fig. 25, showing two more
sheets, st, s5, on another chromosome. Where the spindle tubules enter the chromosomes there are lighter-staining cylindrical regions (arrows), x 21000.
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Fig. 28. Electron micrograph of a section serial to those of Figs. 25, 27, showing the
sheets slt s2 at higher magnification, x 60000.
Fig. 29. Electron micrograph of part of a cell at anaphase containing little or no
haemoglobin. The surfaces of the chromosomes bear numerous projections and some
nuclear envelope-like fragments (arrows). What is presumably endoplasmic reticulum
has ribosomes attached to both sides, x 18000.
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Figs. 30—35. Electron micrographs of six serial sections through a polychromatophil at
anaphase (not the cell shown in Figs. 25, 27, 28), demonstrating the extensive nature of
the envelope-limited sheet (arrows). A second sheet (s) is shown ; the surface of a chromosome has been sectioned at chy. One section (Fig. 30) is stained with uranyl
acetate only; all are x 12000 except Fig. 35, which is x 10000.
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Fig. 36. Electron micrograph of a section serial to that of Fig. 28, showing in the
anaphase chromosome 20-40 A fibrils and granules (arrows), x 240000.
Fig. 37. Electron micrograph of part of a chromosome from a polychromatophil at
prometaphase in which the chromosomal projections (arrows) seen in cross-section
are less well-defined than usual. Ribosomes appear intimately associated with the
chromosomes. Glutaraldehyde (15 %) + OsO 4 . x 32000.
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Fig. 38. Electron micrograph of a cell at anaphase; there are numerous projections
(arrows) on the surfaces of the chromosomes and adjacent blobs caused by sectioning
through projections. A centriole with densely staining surround and a kinetochore
are shown. 1 % osmium tetroxide; methacrylate; lead citrate, x 12000.
Fig. 39. Electron micrograph of a neutrophilic granulocyte in interphase from the
frog {Rana esculenta), showing a sheet (arrow), x 18000.
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Fig. 40. Electron micrograph of a polychromatophil, at the end of anaphase at a stage
corresponding to the light-micrograph, Fig. 10. The chromosomes contain a few large
and many small spaces (arrows). Note the nuclear envelope, and an envelope-limited
sheet with a long discontinuity in the chromatin. x 15 300.
Fig. 41. Electron micrograph of a polychromatophil at telophase. The spaces within
the chromosomes are greatly enlarged (compare with Fig. 40) and contain large
granules (arrows), x 15300.
Fig. 42. Electron micrograph of a polychromatophil at a stage corresponding to that
in the light-micrograph, Fig. 11. What are presumably chromosomes are envelopelimited (arrows), and two are connected by a filament. X4000.
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Fig. 43. Electron micrograph of a polychromatophil at anaphase, the chromosome
bearing an envelope-limited sheet (arrow). 45000.
Fig. 44. Electron micrograph of part of a group of clumped chromosomes, in a polychromatophil at the end of anaphase or early telophase, partially enclosed by nuclear
envelope; note the envelope-limited sheet (arrow), x 13000.
Fig. 45. Electron micrograph of a chromosome from a polychromatophil at prometaphase showing a sheet (arrow), x 40000.
Fig. 46. Electron micrograph of part of a chromosome in the anaphase cell shown in
part in Fig. 35, the section being one from a series. It shows parallel sheets (s,, s2).
x 40000.
Figs. 47, 48. Electron micrographs of two more sections of the anaphase cell of
Fig. 25, showing different aspects of one of the same sheets, s:) (arrows). Fig. 47,
x 36000; Fig. 48, x 30000.
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Fig. 49. Electron micrograph of a nearly mature erythrocyte showing an envelopelimited sheet (arrow) on the surface of the nucleus, x 48000.
Fig. 50. Electron micrograph of a late polychromatophil at interphase showing an
envelope-limited sheet (arrow) in the interior of the nucleus, x 15000.
Fig. 51. Electron micrograph of a late polychromatophil at interphase showing a
sheet (arrows) on the surface of the nucleus, cut in part tangentially with a discontinuity at d. Glutaraldehyde (5 %). x 57000.
Fig. 52. Electron micrograph of a late polychromatophil at interphase, showing two
envelope-limited sheets (arrow) on the surface of the nucleus, x 30000.
Fig. 53. Electron micrograph of a polychromatophil at interphase showing two sheets
(j[, s2); s2 appears to be disconnected from the nucleus, x 45 600.
Fig. 54. Electron micrograph of a polychromatophil at interphase showing an envelope-limited sheet (arrow) protruding from the nucleus, x 45 000.
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