Download nuclear morphology and the ultra

J. CM Sci. 4, 569-582 (1969)
Printed in Great Britain
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NUCLEAR MORPHOLOGY AND THE ULTRASTRUCTURAL LOCALIZATION OF
DEOXYRIBONUCLEIC ACID SYNTHESIS
DURING INTERPHASE
GILLIAN R. MILNER
Department of Medicine, University of Cambridge, England
SUMMARY
The ultrastructural localization of deoxyribonucleic acid (DNA) synthesis was studied by
electron-microscope autoradiography in human transforming lymphocytes, embryonic lung
fibroblasts, epithelial cells and normoblasts. Euchromatin was found to be active in DNA
synthesis in all cell types studied, whereas heterochromatin was inactive. However, D N A
synthesis was also prominent in the regions where heterochromatin was thought to be decondensing to form euchromatin. Analysis of sequential changes in nuclear morphology of the
transforming lymphocyte suggested that there is decondensation of heterochromatin during the
S-phase until none is left. In nuclei with no heterochromatin a prominent localization of DNA
synthesis was at the nuclear membrane. This sequence of complete decondensation of heterochromatin also seemed likely for fibroblasts and epithelial cells. Normoblasts however showed no
stage where the nucleus was wholly euchromatic and it is suggested that in this cell decondensation of heterochromatin for replication is localized and transient.
INTRODUCTION
The cell nucleus possesses chromatin in two forms, a condensed form, heterochromatin, and a less condensed form, euchromatin. The deoxyribonucleic acid
(DNA) concentration of heterochromatin has been shown, by microspectrophotometric measurements of Feulgen-stained nuclei, to be two to three times that of
euchromatin (Lima-de-Faria, 1959). The heterochromatic areas which stain densely
with the uranyl stain used in electron microscopy are identical with those which appear
dense after treatment with the Feulgen stain (Littau, Allfrey, Frenster & Mirsky,
1964).
Autoradiographic studies, with both the light and electron microscopes, suggest
that it is the euchromatic areas which are active in ribonucleic acid (RNA) synthesis
(Littau et al. 1964; Berlowitz, 1965; Karasaki, 1965; Granboulan & Granboulan,
1965) and also in DNA synthesis (Hay & Revel, 1963; Meek & Moses, 1963). Corroboration of these views comes from incorporation studies on nuclei fractionated into
their heterochromatic and euchromatic components (Frenster, Allfrey & Mirsky,
1963)All the DNA of the nucleus must replicate before cell division. If replication occurs
only when the DNA is in the decondensed form, either there must be a stage at which
the nuclear chromatin is totally decondensed, or localized areas of heterochromatin
36
Cell Sci. 4
570
G. R. Milner
must decondense, replicate, and then recondense during the S-phase. Studies of
synchronized cells might be expected to clarify this problem, but it is probable that
the most widely used methods of synchronization by metabolic inhibition affect the
subsequent DNA metabolism of the cell (Till, Whitmore & Gulyas, 1963; Rao &
Engelberg, 1966).
An alternative approach is to follow the changes in the chromatin of lymphocytes
transformed by phytohaemagglutinin where the first interphase is delimited by the
first mitosis. This study has used electron microscopy combined with autoradiography
to follow the changes in nuclear chromatin and sites of DNA synthesis during the first
S-phase of the transforming lymphocyte. A preliminary account of these observations has been published elsewhere (Milner & Hayhoe, 1968). Observations made in
the transforming lymphocyte have now been extended to other cell types.
MATERIALS AND METHODS
Lymphocyte cultures
Lymphocytes from 20 ml of peripheral blood from normal male donors were separated by
sedimentation with gelatin (Coulson & Chalmers, 1964). Cultures were set up in universal
containers in TC 199 containing 200 units/ml penicillin and 100 mg/ml streptomycin and
25 % autologous serum. To each 6-ml aliquot 0-2 ml of phytohaemagglutinin (PHA) was
added. One vial of Bacto-Phytohaemagglutinin M, Difco, was rehydrated with 5 ml of sterile
distilled water. The stationary cultures were incubated with air as the gas phase. At appropriate
intervals tritiated thymidine ([3H]thymidine), specific activity 17-7-22-4 c/mmole (obtained
from the Radiochemical Centre, Amersham) was added to a final concentration of 5 /tc/ml for
specified times. The cultures were then terminated.
Human cell lines in culture
Human embryonic lung fibroblasts were cultured in Eagle's medium with 10% foetal calf
serum and 1 % sodium bicarbonate. They were in their fifth transfer at the time of the experiment. Human epithelial cells originally derived from a laryngeal carcinoma (Moore, Sabachowsky & Toolan, 1955) were grown in TC 199 with 2-5 % foetal calf serum. Both cultures
were kindly supplied by Dr P. Dendy. 5 fic/ml [3H]thymidine were added for 30 min to each
culture and these were then terminated.
Human normoblasts
Fragments of sternal marrow from two normal donors were incubated in TC 199 and [3H]thymidinefor 1 has described above. The fragments were then processed for electron microscopy
and autoradiography.
Light microscopy and autoradiography
Slides for morphology and mitotic counts were stained with Leishman's stain. Slides for
autoradiography were coated by the dipping method with Ilford L4 emulsion after staining
with periodic acid/Schiff stain (PAS). They were exposed for 2 days, and after development
counterstained with haematoxylin.
Electron microscopy and autoradiography
The cultures were terminated byfixationin 6-25 % glutaraldehyde in 015 M phosphate buffer
at pH 7-2 for 1 h. After washing in 0-22 M sucrose in 0-15 M phosphate buffer at pH 7-2 they were
post-fixed in unbuffered 1 % osmium tetroxide for 1 h. Rapid dehydration in a series of alcohols
Ultrastructural sites of DNA synthesis
571
and propylene oxide was followed by embedding in Araldite. Sections were cut on an LKB
Ultrotome, picked up on carbon-coated grids and stained with uranyl acetate. A second thin
carbon film was shadowed on top of the sections and they were then covered with a monolayer
of Ilford L4 emulsion by the loop method (George, 1961). After exposure for 3 weeks, grids
were developed with Microdol-X (Kodak) and further stained with lead citrate (Reynolds,
1963). They were viewed in an AEI EM6B electron microscope.
Whenever cells were counted in the electron microscope care was taken that different thin
sections of the same cell were not included in the count. To ensure this, only one section per
grid was counted and as a routine several thick sections were cut between each successive grid.
RESULTS
In common with other workers (Bender & Prescott, 1962; Mclntyre & Ebaugh,
1962; Michalowski, 1963; Shapiro & Levine, 1967), it was found that there is negligible
DNA synthesis for the first 24 h after initiation of a lymphocyte culture with PHA.
Even at 24 h cells synthesizing DNA were scanty, but by 30 h they averaged 6%.
pHJThymidine added to a culture at this time therefore virtually labelled cells only at
the beginning or in the first part of their 5-phase. The morphology of such cells
showed that [^HJthymidine is taken up only by cells with nuclei containing less
heterochromatin than the parent lymphocyte. Figure 1 shows a cell with the greatest
amount of heterochromatin found. It appears therefore that some decondensation of
chromatin has taken place before the onset of DNA synthesis. It is also apparent from
Fig. 1 that grains appear over areas of euchromatin but there also seems to be a
definite localization at the borders of the heterochromatin. This junctional labelling
was very prominent in some cells and is better seen in Fig. 2. A number of labelled
cells showed the heterochromatin largely confined to a thin band at the periphery of
the nucleus and this was often associated with a considerable concentration of grains.
At 30 h all labelled nuclei contained heterochromatin although in some it was present
merely as a thin marginal rim apposed to the nuclear membrane.
When pHJthymidine was added to a 48-h culture an additional type of cell now
showed labelling. These cells had nuclei with no heterochromatin at all. The time of
appearance of these cells with wholly euchromatic nuclei was investigated and related to other parameters of the lymphocyte cultures. The percentage of transforming
lymphocyte nuclei without any heterochromatin was determined at successive intervals
for cultures from two different donors. Three hundred cells were counted in the
electron microscope for each time interval from each culture. These results were
related to the percentage of cells synthesizing DNA as determined by a count of 500
cells and the mitotic index from a count of 2000 cells from each of 5 cultures. The
percentage of cells in DNA synthesis which had a wholly euchromatic nucleus, as
shown by electron-microscopic autoradiography, is shown for a single culture where
200 labelled nuclei were counted at each time interval. The results are shown in
Table 1.
It can be clearly seen that those cells with a totally decondensed nucleus appeared
after DNA synthesis had begun, but before the stage of mitosis was reached.
The marked localization of grains at the borders of the heterochromatic areas in
some cells was noticeable as also was labelling in the region of the nuclear membrane
36-2
572
G. R. Milner
in cells without heterochromatin. It seemed possible that some of the variation in
patterns of labelling might be due to the length of time for which the label was
available. A io-min pulse of pHJthymidine was therefore given to a 72-h culture of
lymphocytes. Two hundred nuclei were classified as possessing heterochromatin or
being wholly euchromatic. They were further classified as peripheral, if labelling was
confined to the edges of the peripheral heterochromatin or the nuclear membrane if no
heterochromatin was present, as central, if only the central parts of the nucleus were
labelled, and as mixed, if both patterns of labelling were present. Examples are shown
of a cell classified as containing heterochromatin with a mixed pattern of labelling
(Fig. 3) and of a wholly euchromatic nucleus with peripheral labelling (Fig. 4). The
results of this analysis are shown in Table 2.
Table 1. Changes in nuclear morphology, labelling with \^H]thymidine,
and mitotic index in lymphocytes transformed with PHA
Period of culture, h
24
Percentage of:
Nuclei with no heterochromatin
Cells labelled with
[3H]thymidine
Mitoses
Labelled cells with wholly
euchromatic nuclei
30
o
02
o
(0-0-3)
0-4
6
(o— 1) (4-2-9-8)
0 0
—
36
—
166
(12-224)
o
o
42
48
48
(3-3-6-3)
26-2
(23-6-29-4)
0-02
(o-o-i)
8-7
(7-7-9-7)
33-2
(23-6—39-0)
o-6
(0-2-0-9)
8-5
15-0
1-5
Results are expressed as means with the range of values in parentheses.
Table 2. Analysis of nuclear pattern and type of labelling with ^Hjthymidine
for 200 nuclei in a 72-A culture of transformed lymphocytes
Type of labelling
Nuclear morphology
Heterochromatin present
No heterochromatin present
Peripheral
Mixed
Central
68
82
IS
13
IS
7
It can be seen that 91 % of the nuclei containing heterochromatin have labelling
at the border of the heterochromatin and 41 % have labelling at this region only.
Where no heterochromatin is present, the majority of nuclei have labelling at the
nuclear membrane and in 37% this is the sole significant region of labelling.
The finding that there was a period of the 5-phase of the transforming lymphocyte
where the nucleus was of a totally decondensed pattern initiated a search to see if a
similar event also occurred in other cells. In both human embryonic fibroblasts and
human epithelial cells it was found that cells taking up [3H]thymidine showed both
Ultrastructural sites of DNA synthesis
573
the presence and absence of heterochromatin. Labelled nuclei containing no blocks of
heterochromatin from a fibroblast and an epithelial cell are shown in Figs. 5 and 6
respectively. In both types of cell the masses of heterochromatin, where present, were
always small but there did seem to be a relation of grains to these areas suggestive of
the junctional labelling found in the transforming lymphocyte. Normoblasts from
human marrow gave different results: as in the other cell types, grains were confined
to areas of euchromatin (Fig. 7), but a survey of many grids showed no stage, either
labelled or unlabelled, where the nucleus was without substantial masses of heterochromatin.
DISCUSSION
These results confirm previous work which has indicated that the euchromatic
areas of the nucleus are those which are active in DNA synthesis (Hay & Revel, 1963;
Meek & Moses, 1963; Frenster et al. 1963). The improved fixation conferred by
glutaraldehyde has shown that an important site of DNA synthesis is the margin of
the areas of heterochromatin.
No significant labelling over heterochromatin was seen. Where heterochromatin
was present in small masses or in a thin peripheral ring it was not possible to be
certain that grains did not overlie it, but wherever grain size was small, relative to the
areas of heterochromatin, the grains were found to be at the border of the heterochromatin. It seems reasonable to suggest therefore that this is a constant phenomenon.
The change in nuclear morphology with increasing culture time suggests that transformation of the lymphocyte involves gradual decondensation of heterochromatin,
through stages where heterochromatin is present mainly as a diminishing peripheral
rim apposed to the nuclear membrane followed by its eventual disappearance.
Decondensation during the first 24 h is not accompanied by DNA synthesis. Cells
then begin to enter their first 5-phase, the initial part of which at least coincides with
the final stages of decondensation. Areas active in DNA synthesis are those which
are already euchromatic but as new areas of heterochromatin decondense they become
active as a template for DNA synthesis at the point of decondensation.
It might be questioned that some of the cells in DNA synthesis at 48 h are in the
second cell cycle. However in the present cultures mitoses were very scanty before 46 h.
Furthermore, the Gx period in these cells has been estimated as 6 h (Sasaki & Norman,
1966). The majority of cells labelling with [3H]thymidine at 48 h, and in particular the
15 % which have wholly euchromatic nuclei, must therefore be in the 5-phase of the
first cell cycle.
The time in the 5-phase at which the nuclear chromatin becomes totally decondenscd
remains undetermined. It is possible that this is the stage when all the DNA has
replicated. If such is the case the nucleus may or may not retain this morphology
through the G2 rest phase until the condensation of the chromosomes in prophase.
Since it seems that only decondensed chromatin replicates, the alternative, that this
is not the tetraploid stage, might imply further condensation and decondensation
before the end of the S-phase.
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G. R. Milner
This pattern of decondensation of chromatin during interphase may be repeated
during later cell cycles of the transformed lymphocyte. After 72 h incubation, for
example, nuclei with various amounts of heterochromatin are present. Cells in the
iS-phase at 72 h were found to have a similar range of amounts of heterochromatin as
those in the first 5-phase, including cells with no nuclear heterochromatin at all. It
seems probable that heterochromatin could persist from incomplete decondensation
of the chromosomes after telophase as has been found in HeLa cells (Blondel &
Tolmach, 1965).
The complexities of the technique of electron-microscope autoradiography make it
feasible to analyse only a relatively small number of labelled cells and the conclusions
which can be drawn are limited by this fact. Nevertheless it seems probable that the
nuclear membrane is an important locus of DNA synthesis in cells which have no
readily discernible peripheral rim of heterochromatin. This may represent activity
at the end of heterochromatic decondensation as the last remaining layers decondense
at the nuclear membrane. Davies (1968) has recently found that in nuclei from several
species the microfibrils of the heterochromatin lying near the nuclear membrane are
arranged in several ordered layers parallel to the membrane. It seems possible that
the high proportion of euchromatic nuclei showing DNA synthesis in this region
could in part be due to the time taken for linear replication of these microfibrils
parallel to the nuclear membrane. However, the high proportion of wholly euchromatic
nuclei showing labelling in the region of the nuclear membrane suggests that this
region may have special significance.
Evidence that DNA synthesis at membrane surfaces may be of importance comes
largely from bacteria. It has been suggested that the point of replication of the bacterial
chromosome is at the cell membrane (Jacob, Ryter & Cuzin, 1966). It has also been
found that DNA labelled with a short pulse of thymidine sediments with the fraction
of B. subtilis containing the cell membrane (Ganesan & Lederberg, 1965). There have
been suggestions that in higher animals, also, attachment of the chromosomes to a
structure such as the nuclear membrane might explain the phenomenon of segregation
of sister chromatids during mitosis (Lark, Consigli & Minocha, 1966) or the nonrandom arrangement of chromosomes on the metaphase plate (Sved, 1966; Schneidermann & Smith, 1962). Of interest also is the finding in honey-bee embryonic nuclei
that chromatin fibres are attached to the nuclear membrane sometimes in configurations suggesting linear attachment but mainly at the termini of the fibres (DuPraw,
1965)In summary, therefore, the present results suggest that in the transforming lymphocyte active sites of DNA synthesis are in areas of euchromatin and at the borders
of heterochromatic regions as they decondense to become euchromatic. Where no
peripheral heterochromatin lies apposed to the nuclear membrane, this is an important
locus of DNA replication. In some totally euchromatic nuclei the nuclear membrane
region is the sole significant region of such replication; in others more central regions
of euchromatin are also active.
Further work will be necessary to establish if this proposed pattern of ultimately
complete decondensation of heterochromatin during the 5-phase extends to other cell
Ultrastrnctural sites of DNA synthesis
575
types. The rinding of completely euchromatic nuclei during the 5-phase in human
fibroblasts and a human epithelial cell line suggests that it may. On the other hand
Blondel & Tolmach (1965) found no such stage in their study of the ultrastructure
of the nucleus in HeLa cells synchronized by washing off the non-adherent mitotic
cells.
The normoblasts have a nucleus which through several cell divisions becomes gradually more condensed and functionless. Nevertheless it may provide an example of an
alternative pattern. The late normoblast contains the diploid content of DNA (Grasso,
Woodard & Swift, 1963), thus all the DNA must be replicated at each preceding cell
division. Since the present study has indicated that DNA replicates only as euchromatin in this cell type also, and no totally euchromatic nuclei were seen, it seems
probable that local areas of heterochromatin must decondense, replicate and then
condense again during an 5-phase. In keeping with this is the finding that in human
normoblasts there is no relationship between minor differences in nuclear structure and
the position of the cell in interphase as assessed by its DNA content (Wickramsinghe,
Cooper & Chalmers, 1968). Heterochromatin lies apposed to the nuclear membrane
over most of its circumference in this cell type and DNA synthesis has been observed
to be in more centrally lying euchromatic areas. Normoblasts therefore also differ
from transforming lymphocytes in that the region of the nuclear membrane is not a
prominent locus for DNA replication.
The basis for the structural difference between euchromatin and heterochromatin
remains undetermined. It has been shown that demarcation between the types of
chromatin is sharp but there is continuity of microfibrils between them (Frenster,
1965). Removal of the lysine-rich histone fraction from isolated calf thymus nuclei
results in a decrease of density of condensed chromatin, as viewed in the electron
microscope; readdition of this fraction restores the density (Littau, Burdick, Allfrey
& Mirsky, 1965). Furthermore, Whitfield & Perris (1968) have found, also in thymocytes, that both inorganic phosphate and a phosphoprotein cause disappearance of
condensed chromatin which reappears on addition of lysine-rich histones. They have
postulated that phosphate and phosphoprotein combine with histones and thus affect
the interaction of DNA and histone.
The hypothesis that histones act as a mechanism for the repression of DNA has
received much support since its original suggestion (Stedman & Stedman, 1950).
Removal of histone from isolated chromatin increases its activity as a template for
RNA synthesis in vitro (Bonner & Huang, 1963; Allfrey, Littau & Mirsky, 1963;
Georgiev, Ananieva & Kozlov, 1966; Paul, 1967). Alterations in the relationship
between DNA and histone also affect DNA synthesis (Gurley, Irvin & Holbrook,
1964; Billen & Hnilica, 1964; Schwimmer & Bonner, 1965), although the effects are
not as marked as in the case of RNA.
Histones have been implicated in the structural configurations of heterochromatin
(Allfrey et al. 1963) and the chromosomes at cell division (Zubay, 1964). Although
the role of histones in the day-to-day regulation of individual gene function has been
questioned (Paul & Gilmour, 1968) it is possible that histones may provide a mechanism for the more permanent repression of a large part of the genome in a differentiated
576
G. R. Milner
cell (Brown, 1966) It is therefore of interest that the present findings indicate that the
structural change from heterochromatin to euchromatin is accompanied by marked
activity in DNA synthesis.
It has been suggested that chromosomes are located in definite sites in the interphase
nucleus (Mirsky & Osawa, 1961; Comings, 1968) and it is now well established that
replication begins simultaneously at several points along a chromosome and furthermore that the regions of initiation are constant through successive generations (for
example, Taylor, i960; Gilbert, Muldal, Lajtha & Rowley, 1962; Hsu, 1964). The
present demonstration of the important sites of DNA synthesis may provide some
ultrastructural basis for the constancy discernible at the level of the chromosome.
I am grateful to Professor F. G. J. Hayhoe for advice and encouragement and to Mr M. A.
Peacock and Mr R. J. Flemans for expert technical assistance. This work was supported by a
grant from the Medical Research Council to Professor J. S. Mitchell.
REFERENCES
ALLFREY, V. G., LITTAU, V. C. & MIRSKY, A. E. (1963). On the role of histones in regulating
ribonucleic acid synthesis in the cell nucleus. Proc. natn. Acad. Sci. U.S.A. 49, 414-421.
BENDER, M. A. & PRESCOTT, D. M. (1962). DNA synthesis and mitosis in cultures of human
peripheral leukocytes. Expl Cell Res. 27, 221-229.
BERLOWITZ, L. (1965). Correlation of genetic activity, heterochromatization and RNA metabolism. Proc. natn. Acad. Sci. U.S.A. 53, 68-73.
BILLEN, D. & HNILICA, L. S. (1964). Inhibition of DNA synthesis by histones. In The
Nucleohhtones (ed. J. Bonner & P. Ts'o), pp. 289—297. San Francisco: Holden-Day.
BLONDEL, B. & TOLMACH, L. J. (1965). Studies on nuclear fine structure. Expl Cell Res. 37,
497-501.
BONNER, J. & HUANG, R. C. (1963). Properties of chromosomal nucleohistone. J. molec. Biol.
6, 169-174.
BROWN, S. W. (1966). Heterochromatin. Science, N.Y. 151, 417-425.
COMINGS, D. E. (1968). The rationale for an ordered arrangement of chromatin in the interphase nucleus. Am. J. hum. Genet. 20, 440—460.
COULSON, A. S. & CHALMERS, D. G. (1964). Separation of viable lymphocytes from human
blood. Lancet i, 468—469.
DAVIES, H. G. (1968). Electron-microscopic observations on the organization of heterochromatin
in certain cells. J. Cell Sci. 3, 129—150.
DUPRAW, E. J. (1965). The organization of nuclei and chromosomes in honey-bee embryonic
cells. Proc. natn. Acad. Sci. U.S.A. 53, 161-168.
FRENSTER, J. H. (1965). Ultrastructural continuity between active and repressed chromatin.
Nature, Lond. 205, 1341-1342.
FRENSTER, J. H., ALLFREY, V. G. & MIRSKY, A. E. (1963). Repressed and active chromatin,
isolated from interphase lymphocytes. Proc. natn. Acad. Sci. U.S.A. 50, 1026—1032.
GANESAN, A. T. & LEDERBERG, J. (1965). A cell-membrane bound fraction of bacterial DNA.
Biochem. biophys. Res. Commun. 18, 824-835.
GEORGE, L. A. (1961). Electron microscopy and autoradiography. Science, N. Y. 133, 1423-1424.
GEORGIEV, G. P., ANANIEVA, L. N. & KOZLOV, J. V. (1966). Stepwise removal of protein from a
deoxyribonucleoprotein complex and de-repression of the genome. J. molec. Biol. 22,
365-371.
GILBERT, C. W., MULDAL, S., LAJTHA, L. G. & ROWLEY, J. (1962). Time sequence of human
chromosome duplication. Nature, Lond. 195, 869-873.
GRANBOULAN, N. & GRANBOULAN, P. (1965). Cytochimie ultrastructurale du nucleole. Expl
Cell Res. 38, 604-619.
Ultrastructural sites of DNA synthesis
577
GRASSO, J. A., WOODARD, J. W. & SWIFT, H. (1963). Cytochemical studies of nucleic acids and
proteins in erythrocyte development. Proc. natn. Acad. Sci. U.S.A. 50, 134-140.
GURLEY, L. G., IRVIN, J. L. & HOLBROOK, D. J. (1964). Inhibition of DNA polymerase by
histones. Biochem. biophys. Res. Commun. 14, 527—532.
HAY, E. D. & REVEL, J. P. (1963). The fine structure of the DNA component of the nucleus.
J. Cell Biol. 16, 29-51.
Hsu, T. C. (1964). Mammalian chromosomes in vitro. XVIII. DNA replication sequence in
the Chinese hamster. J . Cell Biol. 23, 53-62.
JACOB, F., RYTER, A. & CUZIN, F. (1966). On the association between DNA and membrane in
bacteria. Proc. R. Soc. B 164, 267-278.
KARASAKI, S. (1965). Electron microscopic examination of the sites of nuclear RNA synthesis
during amphibian embryogenesis. J. Cell Biol. 26, 937—958.
LARK, K. G., CONSIGLI, R. A. & MINOCHA, H. C. (1966). Segregation of sister chromatids in
mammalian cells. Science, N.Y. 154, 1202-1205.
LIMA-DE-FARIA, A. (1959). Differential uptake of tritiated thymidine into hetero- and euchromatin in Melanoplus and Secale. J. biophys. biochem. Cytol. 6, 457-466.
LITTAU, V. C , ALLFREY, V. G., FRENSTER, J. H. & MIRSKY, A. E. (1964). Active and inactive
regions of nuclear chromatin as revealed by electron microscope autoradiography. Proc.
natn. Acad. Sci. U.S.A. 52, 93-100.
LITTAU, V. C , BURDICK, C. J., ALLFREY, V. G. & MIRSKY, A. E. (1965). The role of histones
in the maintenance of chromatin structure. Proc. natn. Acad. Sci. U.S.A. 54, 1204-1212.
MCINTYRE, O. R. & EBAUGH, F. G. JR. (1962). The effect of phytohaemagglutinin on leukocyte cultures as measured by P32 incorporation in the DNA, RNA, and acid soluble fractions.
Blood 19, 443-453.
MEEK, G. A. & MOSES, M . J . (1963). Localisation of 3 H-thymidine in HeLa cells. Jl R.
microsc. Soc. 81, 187—197.
MICHALOWSKI, A. (1963). Time course of DNA synthesis in human leukocyte cultures. Expl
Cell Res. 32, 609-612.
MILNER, G. R. & HAYHOE, F. G. J. (1968). Ultrastructural localization of nucleic acid synthesis
in human blood cells. Nature, Lond. 218, 785-787.
MIRSKY, A. E. & OSAWA, S. (1961). In The Cell, vol. 2 (ed. J. Brachet & A. E. Mirsky),
p. 694. New York and London: Academic Press.
MOORE, A. E., SABACHOWSKY, L. & TOOLAN, H. W. (1955). Culture characteristics of four
permanent cell lines of human cancer cells. Cancer Res. 15, 598—602.
PAUL, J. (1967). Masking of genes in cytodifferentiation and carcinogenesis. In Cell Differentiation, A CIBA Fdn Symp. (ed. A. V. S. de Reuck & J. Knight), pp. 196-202. London:
Churchill.
PAUL, J. & GILMOUR, R. S. (1968). Organ-specific restriction of transcription in mammalian
chromatin. J. molec. Biol. 34, 305-316.
RAO, P. N. & ENCELBERG, J. (1966). Effects of temperature on the mitotic cycle of normal and
synchronised mammalian cells. In Cell Synchrony (ed. I. V. Cameron & G. M. Padilla),
pp. 332-352. New York: Academic Press.
REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in
electron microscopy. J. Cell Biol. 17, 208-212.
SASAKI, M. S. & NORMAN, A. (1966). Proliferation of human lymphocytes in culture. Nature,
Lond. 210, 913-914.
SCHNEIDERMAN, L. J. & SMITH, C. A. B. (1962). Non-random distribution of certain homologous pairs of normal human chromosomes in metaphase. Nature, Lond. 195, 1229-1230.
SCHWIMMER, S. & BONNER, J. (1965). Nucleohistone as template for the replication of DNA.
Biochim. biophys. Acta 108, 67-72.
SHAPIRO, I. M. & LEVINE, L. Y. (1967). Autoradiographical study on the time of nuclear
protein synthesis in human leucocyte blood culture. Expl Cell Res. 47, 75-85.
STEDMAN, E. & STEDMAN, E. (1950). Cell specificity of histones. Nature, Lond. 166, 780-781.
SVED, J. A. (1966). Telomere attachment of chromosomes: some genetical and cytological
consequences. Genetics 53, 747-756.
TAYLOR, J. H. (i960). Asynchronous duplication of chromosomes in cultured cells of Chinese
hamster. J. biophys. biochem. Cytol. 7, 455-463.
578
G. R. Milner
J. E., WHITMORE, G. F. & GULYAS, S. (1963). Deoxyribonucleic acid synthesis in individual L-strain mouse cells. II. Effects of thymidine starvation. Biochim. biophys. Acta 72,
277-289.
WHITFIELD, J. F. & PERRIS, A. D. (1968). Dissolution of the condensed chromatin structures of
isolated thymocyte nuclei and the disruption of deoxyribonucleoprotein by inorganic phosphate and a phosphoprotein. Expl Cell Res. 49, 359-372.
WICKRAMSINGHE, S. N., COOPER, E. H. & CHALMERS, D. G. (1968). A study of erythropoiesis
by combined morphologic, quantitative, cytochemical and autoradiographic methods.
Blood 31, 304-313.
ZUBAY, G. (1964). Nucleohistone structure and function. In The Nucleohistones (ed. J. Bonner
& P. Ts'o), pp. 95-107. San Francisco: Holden-Day.
TILL,
(Received 6 August 1968)
Figs. 1, 2. Electron-microscope autoradiographs of transforming lymphocytes from a
30-h culture labelled with fHJthymidine. Grains are seen in areas of euchromatin
and where heterochromatin borders on to euchromatin (nu, nucleolus). Fig. 1, x 18000;
Fig. 2, x 25000.
Vltrastructural sites of DNA synthesis
579
G. R. Milner
Figs. 3, 4. Electron-microscope autoradiographs of transforming lymphocytes from a
72-h culture labelled with [8H]thymidine. The cell in Fig. 3 was classified as possessing
heterochromatin with a mixed pattern of labelling, x 19000. The cell in Fig. 4 was
classified as being euchromatic with peripheral labelling (nil, nucleolus). x 23000.
Ultrastructural sites of DNA synthesis
Figs. 5, 6. Electron-microscope autoradiographs of nuclei which possess no blocks
of heterochromatin labelled with ['HJthymidine (mi, nucleolus). Fig. 5. Nucleus of
a human embryonic fibroblast. x 12000. Fig. 6. Nucleus of a human epithelial cell
nucleus, x 12000.
G. R. Milner
Fig. 7. Electron-microscope autoradiograph of two human normoblasts labelled with
[3H]thymidine. Grains are localized solely over areas of euchromatin. x 18000.