/. Embryol. exp. Morph. Vol. 55, pp. 183-194, 1980
Printed in Great Britain © Company of Biologists Limited 1980
\ 33
Drosophila nuclei replicate in Xenopus eggs
By CHRISTINA SMITH 1 , DAVID BRILL 1 , MARY BOWNES 2 AND
CHRISTOPHER FORD 1
From the School of Biological Sciences, University of Sussex
SUMMARY
Nuclei isolated from a permanent cell line derived from Drosophila melanogaster embryos
have been injected, along with a radioactive DNA precursor [3H]TTP, into Xenopus laevis
eggs. In culture, less than 7 % of the cells were in S phase. After a 90 min incubation, following
injection into eggs, 99% of the nuclei were shown by autoradiography to have synthesized
DNA. In a similar experiment, a density label BrdUTP was injected into eggs along with the
nuclei. Subsequent analysis on caesium chloride gradients showed that this DNA synthesis
was semi-conservative replication. Therefore we conclude that signals present in Xenopus
egg cytoplasm can initiate and sustain true semi-conservative DNA replication in nuclei
from an invertebrate organism.
INTRODUCTION
Following fertilization, an amphibian embryo is primarily concerned with
multiplying from 1 cell to 10000 cells or more without growth, to obtain the
necessary numbers of cells for morphogenetic movements. Such a multiplication
requires that the early embryo undergoes two main processes: DNA replication
and mitosis. To achieve this quickly, an unfertilized egg has stored sufficient
components to sustain many rounds of DNA replication without further synthesis of precursors (see, for example, Benbow, Pestell & Ford, 1975). Since a
unfertilized egg is large and easily manipulated, it is a good experimental system
for studying the mechanism of DNA replication.
In addition, there is a critical difference between oocytes and eggs. An oocyte,
although containing major components for DNA replication like polymerases,
is unable to initate DNA synthesis in injected Xenopus Gx nuclei (Gurdon, 1967).
However, after maturation of these oocytes by hormone treatment, the now unfertilized egg can induce Xenopus Gx nuclei (e.g. brain, liver, blood) to enter S
phase (Graham, Arms & Gurdon, 1966).
The action of injecting nuclei into unfertilized eggs artificially promotes
development, and the eggs procede through the first cell cycle of embryogenesis.
1
Authors' address: School of Biological Sciences, University of Sussex, Falmer, Brighton,
BN1 9QG, U.K.
2
Authors' address: Department of Biology, University of Essex, Wivenhoe Park, Colchester,
CO4 3SQ, U.K.
184
C. SMITH, D. BRILL, M. BOWNES AND C. FORD
Injected sperm nuclei, at least, behave in the same manner as the host egg nucleus; that is, they swell during the first 20 min of the cell cycle, synthesize DNA
during the next 20 min and stop synthesis in the G2 phase of the cycle (Graham,
1966). Therefore, it seems likely that injected nuclei are under the control of
cytoplasmic signals present in the egg.
Heterologous nuclei have also been injected into Xenopus eggs. Both mouse
liver (Graham et al. 1966) and Hela (de Roeper, Smith, Watt & Barry, 1977)
nuclei respond to egg signals and are induced to synthesize DNA. This implies
that some initiation factors involved in DNA synthesis are not species specific
and may be general among vertebrates. Studies on cell fusion between erythrocyte nuclei and mouse fibroblast cells also demonstrate this point (see
Harris, 1968).
DNA synthesis, in injection experiments, was detected by auto radiography
after injection of a radioactive DNA precursor along with the nuclei. It has been
assumed that such autoradiographic labelling represented DNA replication
and not some form of repair. In the case of homologous nuclei this assumption is
indirectly supported by nuclear transplantation experiments. When a single
Xenopus somatic cell nucleus is injected as a distorted cell into an ultravioletirradiated egg, a normal embryo can result (see Gurdon & Laskey, 1970).
Clearly, in this case, initiation and propagation of DNA replication is occurring
in a correct manner. Laskey & Gurdon (1973) have also demonstrated semiconservative DNA synthesis of polyoma DNA injected into eggs. However, it
may not be valid to extend these conclusions to experiments in which large
numbers of Gx nuclei are injected. These nuclei are isolated in sucrose-Mg2+
solutions, and it is known that such isolation procedures introduce many nicks
into the DNA (Hewish & Burgoyne, 1973; Halldorssen, Gray & Shall, 1978).
Therefore, the autoradiographic labelling of these nuclei may be a consequence
of repair of the damaged DNA.
In this paper, we further explore the concept that initiation of DNA replication is a universal process by injecting nuclei from an invertebrate source,
Drosophila melanogaster, into Xenopus laevis eggs. Such nuclei do initiate DNA
synthesis in response to egg signals. In addition, we show that this DNA synthesis, detected by autoradiography and by isolation of radioactive DNA on
caesium chloride gradients, is semi-conservative replication.
MATERIALS AND METHODS
Chemicals
Lysolecithin, grade I and Bovine albumen fraction V were obtained from
Sigma. Radioactive compounds were obtained from the Radiochemical Centre,
Amersham. 5-Bromo-2'-deoxyuridine 5'-triphosphate was obtained from the
Boehringer Corporation and CsCl ('analar' grade) from BDH.
Drosophila nuclei in Xenopus
185
Living materials
Xenopus laevis females were maintained and induced to lay eggs as described
by Gurdon (19676). Unfertilized eggs were collected in modified Barth solution
containing 115 mM NaCl (high salt Barth X), dejellied by swirling in 2%
cysteine pH 8-0 and then maintained before injection in high-salt Barth X.
Drosophila melanogaster. Cultured cell line D x (Dubendorfer & Shields, 1972)
was derived from wild-type Drosophila embryos and subcultured every seven days
in a medium described by Shields & Sang (1977). Cells were grown to confluence
in Carrell flasks. At confluence the cells have stopped cycling and can be shaken
from the flask as a single cell suspension for nuclear isolation and subculturing.
Oregon R adults were maintained on an agar yeast, cornmeal and sugar
medium at 25 °C. Eggs were collected for a 1 h period and the larvae grown on
medium well seeded with live yeast.
Preparation of nuclei
(1) From cultured cells. 0-3 ml of detached cells containing 2-0 x 106 cells
were centrifuged for 5 min at 2500 rev./min in a Mistral 2L. The supernatant
was decanted and the pellet resuspended in 0-3 ml SUNAP solution (0-25 M
sucrose, 75 mM NaCl, 0-15 mM spermine, 0-5 mM spermidine (Gurdon, 1976)
10 jiil of lysolecithin (1 mg/ml) was added at room temperature. After shaking
for 90 sec, cell lysis was stopped by adding 1 ml of ice-cold SUNAP-BSA
(SUNAP with 3% w/v BSA). Nuclei were pelleted by centrifuging for 2 min
at 2000 rev./min in a Mistral 2L and resuspended in 50 y\ SUNAP-BSA for
injection.
(2) From Drosophila larval imaginal discs. The imaginal discs from 40 late
instar larvae were dissected into Drosophila Ringers (Chan & Gehring, 1971).
They were transferred to SUNAP-BSA in a 0-1 ml Jencon's homogenizer (Cat.
no. H103/33) and homogenized with two slow up-and-down strokes. This make
of homogenizer disrupts 90% of the cells to release intact nuclei. The nuclei
were pelleted by centrifuging at 2000 rev./min for 2 min and resuspended in
50/4 of SUNAP-BSA.
Solutions for injection
For autoradiographic analysis eggs were injected with 40 nl of solution which
was prepared by adding 10/d of nuclear suspension to 40/*Ci [3H]TTP (Amersham, specific activity 30 Ci/m mole) previously dried under vacuum. For
density substitution experiments equal volumes of BrdUTP (20 mM in SUNAPBSA) and nuclei were mixed and 10 JLL\ of this added to 40 /*Ci of [3H]dGTP
(specific activity 12-4 Ci/m mole) previously dried under vacuum. Some of the
eggs injected with BrdUTP and [3H]dGTP were also prepared for autoradiography.
186
C. SMITH, D. BRILL, M. BOWNES AND C. FORD
Histology and autoradiography
Injected eggs for autoradiographic analysis were incubated at 21 °C for 90
min in modified Barth's solution. They were fixed in Bouin's solution, embedded
in wax and sectioned at 8 fim. Sections were stained in Mayer's haemalum and
light green. Slides were dipped in Ilford K2 emulsion and, after exposure for 5
or 7 days at 4 °C, developed using PQ universal developer, restained and
scored.
Caesium chloride centrifugation
Injected eggs in density substitution experiments were incubated at 21 °C for
up to 6 h and then frozen at — 20 °C. DNA was extracted as described by Ford
& Woodland (1975) with the following modifications. All extractions were performed under minimal lighting in tubes covered with silver foil. Samples were
homogenized in a final volume of 4 ml of 100 mM Tris-HCl, 50 mM NaCl, 10 mM
disodium EDTA, and then incubated at 37 °C with ribonuclease (20 /tg/ml final
concentration) for 30 min, followed by pronase (500/tg/mlfinalconcentration) for
2 h. Samples were extracted twice with chloroform isoamyl alcohol (24:1, v/v)
and the aqueous phase loaded directly onto CsCl gradients. CsCl gradients were
prepared by adding solid CsCl to extracted samples to give a final volume of
6 ml and a final density of 1-74 g/cm3. Samples were centrifuged for 60 h at
40000 rev./min in a Beckman Ti 50 rotor, and fractions collected and analysed
as described by Ford & Woodland (1975).
To determine whether any of the [3H]dGTP-labelled nucleic acid synthesized
in eggs was RNA, some batches were homogenized as indicated above, digested
with pronase, and extracted twice with phenol and then ether. Aliquots were
then digested with preboiled ribonuclease (40/tg/ml final concentration) or
deoxyribonuclease (200 /tg/ml final concentration, in the presence of 5 mM
MgCl2 in excess of EDTA). Samples were precipitated with 5% TCA, filtered
and counted. 94% of control levels of acid-insoluble counts were recovered after
ribonuclease treatment, and 7% after deoxyribonuclease digestion.
RESULTS
The purpose of this study was to determine whether nuclei from an invertebrate source, Drosophila melanogaster, would respond to cytoplasmic signals of
vertebrate origin and initiate DNA replication. The cells chosen for this were a
cultured cell line D, which are primarily diploid and have a generation time of
about 24 h. These cells are particularly useful since at confluence the cells spontaneously lift off the dish to give a single-cell suspension convenient for nuclear
isolations. These suspended cells also remain viable, and are used for routine
subculturing.
Nuclei were isolated from cell suspensions derived from confluent cultures
Drosophila nuclei in Xenopus
187
Fig. ]. Nuclear swelling and autoradiographic labelling in Drosophila nuclei ninety minutes
after injection into eggs, {a) Swollen disc nuclei. (6) Swollen disc nucleus, (c) Autoradiograph
of (6). (d) Swollen cultured cell nuclei, (e) Swollen cultured cell nuclei. (/) Autoradiograph of
(e).
188
C. SMITH, D. BRILL, M. BOWNES AND C. FORD
Table 1. DNA synthesis in Drosophila nuclei injected into Xenopus eggs
Eggs and oocytes were injected with nuclei and [3H]TTP, incubated for 90 minutes
and then examined by autoradiography. After 7 days exposure, labelled nuclei had
more than 20 grains per nucleus above a background which was never more than 3
grains measured on an equivalent area of cytoplasm.
Nuclei
Exposure
time
Female no. (days)
7
Imaginal discs
1
2
3
4*
5
Cultured cell
6
7
Cultured cell
7
7
5
7
No. of
eggs
scored
Nuclei scored
r
Labelled
Proportion
Unlabelled* labelled
4
3
6
395
248
185
1
0
3
7
291
0
8
88
8
99-8
100
98-4
100
91-7
6t
0
69
0
3
* Eggs from female 4 were injected with nuclei, BrdUTP and. [ H]dGTP and then incubated
and examined by autoradiography in the same way.
t Oocytes.
using a method described by Gurdon (1976), see Methods. The proportion of
whole cells remaining in the final nuclei preparation was determined by dye
exclusion using trypan blue. Each nuclear preparation used for injection contained less than 15% whole cells.
The nuclear suspension was injected into eggs together with [3H]TTP. By 90
min of incubation the injected nuclei were swollen and radioactively labelled
(Fig. 1). The results of three experiments using eggs from different frogs (Table
1) showed that almost every nucleus was autoradiographically labelled. Similarly, nuclei injected together with BrdUTP and [3H]dGTP were all labelled
autoradiographically (Table 1). In this case [3H]dGTP-labelled nucleic acid
extracted from parallel batches of eggs was sensitive to deoxyribonuclease, but
insensitive to ribonuclease (see Methods). It is concluded that the vast majority
of the radioactive material detected autoradiographically is DNA.
To eliminate the possibility that Drosophila Gx nuclei contain their own
endogenous initiation signals which are simply activated upon injection into
eggs, they have also been injected into oocytes. In this case, no incorporation of
radioactivity was detected (Table 1).
The interpretation of these results depends upon what proportion of the cells
used for nuclear isolation were synthesizing DNA at the time of isolation. This
was determined for each experiment by incubating confluent cultures, with [3H]thymidine (5 /tCi/ml) for 2 h. The cells were pelleted, spread on slides and autoradiographed. Figure 2 shows the distribution of labelling of the confluent
Drosophila cells in one of these experiments. The proportion of cells synthesizing DNA at the time of isolation was thus at most 7% in this experiment. In
Drosophila nuclei in Xenopus
189
80-
60-
40-
20-
0-4
5-9
10-14
15—19
Grains per nucleus
20 +
Fig. 2. Labelling index of confluent Drosophila cells. The total number of grains per
nucleus was scored for 941 nuclei. Background varied in different parts of the slides
from 0 to 3 grains per equivalent nuclear area.
every experiment the labelling index of these cultures was never more than
10%.
Thus the majority of cells taken from the confluent cultures was not synthesizing DNA. Therefore the observation that nearly all Drosophila nuclei
synthesized DNA after injection into eggs, but not into oocytes, implies that the
induction of this synthesis is controlled by initiation signals present in Xenopus
eggs.
It is thus shown that nuclei from Drosophila cultured cells do respond to
vertebrate initiation signals. Since the cultured cells are a permanent cell line
and may have altered control mechanisms, nuclei have also been isolated from
third instar larval imaginal discs. Although only small numbers of disc nuclei
were injected into each egg it is clear that a high proportion of these nuclei are
also induced to synthesize DNA within 90 min (Table 1). Incubation of whole
discs in [3H]TdR (10/tCi/ml) for 90 min indicated that only 5% of these cells
were synthesizing DNA at the time of isolation. Therefore nuclei isolated directly
from larval cells as well as nuclei from cultured cells respond to signals to initiate
DNA synthesis in Xenopus egg cytoplasm.
To determine whether the DNA synthesis induced in Drosophila nuclei was
semi-conservative replication, eggs were injected with 10 mM BrdUTP as well as
nuclei and [3H]dGTP in order to density label the newly synthesized DNA.
13
EMB 55
190
C. SMITH, D. BRILL, M. BOWNES AND C. FORD
2-
1-
u
4-
3-
2-
1-
-0-6
20
Fraction number
40
Fig. 3. CsCl gradient analysis of labelled DNA isolated from injected eggs. A.
Control sample of 33 eggs injected with [3H]dGTP and BrdUTP and incubated for
5 h. B. DNA extracted from 38 eggs injected with Drosophila nuclei as well as [3H]dGTP and BrdUTP and incubated for 5 h. Calf thymus DNA was added to each
sample to provide an optical density marker. Recovery of this DNA through the
extraction procedure was 77 % and 65 % for A and B respectively.
DNA was extracted from batches of eggs and analysed by equilibrium CsCl
gradient centrifugation. As a control, a batch of eggs was injected with BrdUTP
and [3H]dGTP only. The radioactive DNA appeared as a single peak about
70 mg/ml denser than unsubstituted marker DNA (Fig. 3d). Complete substitution of BrdUTP for TTP in one strand would produce only a 50 mg/ml
shift in Xenopus DNA (which has a G + C content of 40%). Therefore the
Drosophila nuclei in Xenopus
191
Table 2. Percentage radioactivity in density-substituted regions of
CsCl gradients
Results using eggs from two females are given. The amount of DNA synthesized
(column 5) was calculated as described by Ford & Woodland (1975) and assumed that
all the [3H]dGTP injected was present as triphosphate. The endogenous dGTP pool
was assumed to be 10 moles per egg (Woodland & Pestell, 1972). The radioactivity
present in the density-shifted part of the gradients was adjusted for recovery before
calculating the proportion of dGTP pool incorporated. dGTP was assumed to have
been incorporated in proportion to the G + C content of Drosophila DNA (taken as
40%).
Incubation
time (h)
3£
4
Nuclei
injected
None
None
None
5
3
Drosophila
5
Drosophila
6
Drosophila
% Radioactivity in
DNA syntheHH (1 -770 g/ml) HL (1 -735 g/ml) sized (pg/egg)
96
95
94
100
59
45
65
59
78
4
5
6
0
41
55
35
41
22
13
24
23
20
42
41
99
46
.147
observed shift of 70mg/ml reflects partial substitution in both DNA strands.
Semi-conservative replication of Xenopus egg chromosomal DNA was detected
in this experiment because the eggs were not irradiated prior to injection, and
consequently the undamaged egg nucleus underwent several rounds of replication after activation (Ford & Woodland, 1975). Replication of the egg nuclear
DNA therefore provided an internal marker for the degree of substitution in this
experiment. In addition, the unsubstituted density of Xenopus DNA (1-699
g/ml, Dawid, 1965) and Drosophila DNA (1-702 g/ml quoted by Berendes,
1973) were indistinguishable under these centrifugation conditions. Replication
of Drosophila DNA in Xenopus eggs should therefore produce radioactive bands
at about 1-735 g/ml and 1-770 g/ml for heavy-light (HL) and heavy-heavy
(HH) DNA respectively.
Figure 3 b shows the result obtained when Drosophila nuclei were injected into
eggs with BrdUTP and [3H]dGTP. There is now a new major peak of radioactivity at 1-735 g/ml. This indicates that the Drosophila DNA has undergone
semi-conservative replication. The data obtained from CsCl analysis of batches
of eggs incubated for different durations is summarized in Table 2. In all cases
where Drosophila nuclei were injected, a large peak of radioactivity was observed
in the HL region. Even by 3 h of incubation the Drosophila nuclei have undergone extensive first-round semi-conservative replication.
The radioactivity in both the HL and the HH peaks in fact represents equiva13-2
192
C. SMITH, D. BRILL, M. BOWNES AND C. FORD
40
50
Fraction
Fig. 4. CsCl gradient analysis of alkali-denatured DNA. Thirty eggs were injected
with Drosophila nuclei, [3H]dGTP and BrdUTP and incubated for 5 h. After DNA
extraction the sample (4-3 ml) was denatured by adding 0-1 ml 5 M-NaOH, and then
neutralized 5 min later by adding 0-1 ml 5 M-HCI. The sample was then analysed on
a neutral CsCl gradient as described in the Methods.
lent levels of substitution in one and both strands respectively, because a single
density-shifted peak is observed on CsCl after alkali denaturation (Figure 4).
Some of the Drosophila DNA may have undergone a second round of replication after 5 or 6 h incubation because there is a large increase in the size of the
HH peak compared to the controls (Figure 3 and Table 2). Injection of Drosophila nuclei stimulates the level of DNA synthesis two to five times compared to
controls (Table 2, column 5). This increased synthesis is primarily accounted for
at early times by the large increase in the HL peak. At later times both of the
density-substituted peaks are increased. These data may indicate that some
Drosophila nuclei complete one round of replication within 3 ^ h of injection
and that these nuclei enter a second round of replication within 5 h.
The combined data from autoradiograph and CsCl analysis indicate that the
stimulated level of DNA synthesis observed in the presence of Drosophila nuclei
is due to replication of Drosophila DNA. Two possible alternative interpretations
can be excluded. First, the injection of Drosophila nuclei could stimulate host
nuclear DNA synthesis. However, Drosophila nuclei were induced to synthesize DNA in the presence of BrdUTP and [3H]dGTP (Table 1). In addition, some
20-100 nuclei were scored in each egg, of which two could have been host nuclei.
Secondly, injection of Drosophila nuclei may stimulate DNA synthesis of
Drosophila nuclei in Xenopus
193
Xenopus amplified rDNA. In this case the radioactive peak at density 1-733
(Fig. 3 b) would be rDNA labelled by repair synthesis, and the peak at 1-767 may
contain rDNA that has undergone semi-conservative replication. However, the
presence of a single peak at a density of 1-784 on CsCl after alkali denaturation
(Fig. 4) is not consistent with either replication or repair synthesis of the amplified rDNA .
DISCUSSION
A high proportion of Drosophila nuclei isolated either from stationary-phase
cultured cells or late third instar imaginal discs initiate DNA synthesis within
90 min of injection into Xenopus eggs. The DNA synthesis detected autoradiographically was shown to be semi-conservative replication because BrdUTP was
incorporated giving discrete HL and HH peaks on CsCl gradients. Components
in Xenopus eggs are therefore capable of initiating and continuing replication of
DNA from an invertebrate source. These observations are consistent with the
view that signals initiating replication in invertebrates are similar to those in
vertebrates. However, it is not known whether the replication of Drosophila
nuclei in Xenopus eggs is initiated at replicon initiation sites normally active in
Drosophila cells. If replicon initiation sites are short DNA sequences, but are
different in Xenopus and Drosophila, it is possible that the Xenopus sequence
occurs by chance in the Drosophila genome and is used for initiating replication
by the Xenopus machinery.
It is clear from the density-substitution results that repair synthesis, if it
occurs at all in this situation, is a trivial proportion of the total synthesis that
occurs. Nuclei isolated in sucrose-Mg2+ solutions can have nicks introduced
into the DNA (Hewish & Burgoyne, 1973; Halldorsson et al. 1978). Fewer nicks
are introduced when spermidine and spermine are used instead of Mg2+ to
stabilize the nuclei. Whatever the number of nicks introduced during nuclear isolation in the present study, the nicks do not stimulate significant repair synthesis
compared to the amount of semi-conservative replication that occurs following
injection. Induction of replication is reflected therefore in the increase in the proportion of nuclei that are labelled autoradiographically. The amount of DNA
synthesis in eggs has been estimated from a knowledge of the pool sizes of the
deoxynucleoside triphosphates in eggs (Woodland & Pestell, 1972), the amount
of radioactivity injected and the amount of radioactivity incorporated into
DNA banded on CsCl (Table 2). In similar experiments by Ford & Woodland
(1975) it was shown that during incubation up to 2 h, the host nucleus synthesized an amount of DNA which could be predicted from the cell cycle times of
haploid embryos. This suggests that the calculated amounts of synthesis are
accurate at least within a factor of two. From these estimates the amount of
synthesis stimulated by Drosophila nuclei is 10-20 pg at 3-4 h and between 20
and 120 pg at 5-6 h. A diploid Drosophila nucleus contains 0-36 pg DNA
(Rasch, Barr & Rasch, 1971). Therefore the equivalent of about 100 Drosophila
194
C. SMITH, D. BRILL, M. BOWNES AND C. FORD
nuclei was entirely replicated during 3-4 h. In these experiments a maximum of
about 100 nuclei per egg was detected histologically. This quantitation of DNA
synthesis supports the conclusion based on density substitution data that most
Drosophila nuclei complete one round of replication within 3-4 h and have
entered a second round by 5 h.
We are most grateful to Professor J. H. Sang and Linda Fisher for providing Drosophila
cells. One of us (M.B.) thanks the Science Research Council for travel support. We especially
thank the Cancer Research Campaign for grants supporting this work.
REFERENCES
R. M., PESTELL, R. Q. W. & FORD, C. C. (1975). Appearance of DNA polymerase
activities during early development in Xenopus laevis. Devi, Biol. 43, 159-174.
BERENDES, H. D. (1973). Synthetic activity of polytene chromosomes. Int. Rev. Cytol. 35,
61-116.
BROWN, D. D. & LITTNA, E. (1964). Variations in the synthesis of stable RNX's during
oogenesis and development, /. molec. Biol. 8, 688-695.
CHAN, L. N. & GEHRING, W. (1971). Determination of blastoderm cells in Drosophila
melanogaster. Proc. natn. Acad. Sci., U.S.A. 68, 2217-2221.
DAWID, I. B. (1965). Deoxyribonucleic acid in Amphibian eggs. /. molec. Biol. 12, 581-599.
BENBOW,
de ROEPER, A., SMITH, J. A., WATT, R. A. & BARRY, J. M. (1977). Chromatin dispersal and
DNA synthesis in Gx and G2 Hela cell nuclei injected into Xenopus eggs. Nature, Lond.
265, 469-470.
DUBENDORFER, A. & SHIELDS, G. (1972). Proliferation in vitro and in vivo of a cell line
originally derived from imaginal disc cells. Drosphila Information Service 49, 43.
FORD, C. C. & WOODLAND, H. R. (1975). DNA synthesis in oocytes and eggs of Xenopus laevis
injected with DNA. Devi Biol. 43, 189-199.
GRAHAM, C. F. (1966). The regulation of DNA synthesis and mitosis in multinucleate frog
eggs. / . Cell. Sci. 1, 363-374.
GRAHAM, C. F., ARMS, K. & GURDON, J. B. (1966). The induction of DNA synthesis by frog
egg cytoplasm. Devi Biol. 14, 439-460.
GURDON, J. B. (1967). On the origin and persistence of a cytoplasmic state inducing nuclear
DNA synthesis in frogs' eggs. Proc. natn. Acad. Sci., U.S.A. 58, 545-552.
GURDON, J. B. (19676). 'African Clawed Frogs'. In Methods in Developmental Biology (ed.
F. H. Wilt & N. K. Wessels), pp. 75-84. Thos. Crowell Co., N.Y.
GURDON, J. B. (1976). Injected nuclei in frog oocytes: fate, enlargement and chromatin
dispersal. / . Embryol. exp. Morph. 36, 523-540.
GURDON, J. B. & LASKEY, R. A. (1970). The transplantation of nuclei from single cultured
cells into enucleate frogs' eggs. /. Embryol. exp. Morph. 24, 227-247.
HALLDORSSEN, H., GRAY, D. A. & SHALL, S. (1978). Poly (ADP-Ribose) polymerase activity
in nucleotide permeable cells. FEBS Letters 85, 349-352.
HARRIS, H. (1968). Nucleus and Cytoplasm, p. 100. Oxford: Clarendon Press.
HEWISH, D. R. & BURGOYNE, L. A. (1973). The calcium dependent endonuclease activity of
isolated nuclear preparations. Relationships between its occurrence and the occurrence of
other classes of enzymes found in nuclear preparations. Biochemical and Biophysical
Research Communications 52, 475-481.
LASKEY, R. A. & GURDON, J. B. (1973). Induction of Polyoma DNA synthesis by injection
into frog-egg cytoplasm. Eur. J. Biochem. 37, 467-471.
RASCH, E. M., BARR, H. J. & RASCH, R. W. (1971). The DNA content of sperm of D. melanogaster. Chromosoma 33, 1-18.
SHIELDS, G. & SANG, J. H. (1977). Improved medium for culture of Drosophila embryonic
cells. Drosophila Information Service 52, 161.
WOODLAND, H. R. & PESTELL, R. Q. W. (1972). Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis. Biochem. J. 127, 597-605.
(Received 23 January 1979, revised 7 September 1979)