sign in sign up
<<
Home , Drosophila, Drosophila melanogaster, Xenopus

/. Embryol. exp. Morph. Vol. 55, pp. 183-194, 1980 \ 33 Printed in Great Britain © Company of Biologists Limited 1980

Drosophila nuclei replicate in eggs

By CHRISTINA SMITH1, DAVID BRILL1, MARY BOWNES2 AND CHRISTOPHER FORD1 From the School of Biological Sciences, University of Sussex

SUMMARY Nuclei isolated from a permanent cell line derived from 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 can initiate and sustain true semi-conservative DNA replication in nuclei from an .

INTRODUCTION Following fertilization, an 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 . To achieve this quickly, an unfertilized egg has stored sufficient components to sustain many rounds of DNA replication without further syn- thesis 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 and eggs. An , 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 un- fertilized egg can induce Xenopus Gx nuclei (e.g. , 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 of embryogenesis. 1 Authors' address: School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, U.K. 2 Authors' address: Department of , University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, U.K. 184 C. SMITH, D. BRILL, M. BOWNES AND C. FORD Injected nuclei, at least, behave in the same manner as the host egg nuc- leus; that is, they swell during the first 20 min of the , synthesize DNA during the next 20 min and stop synthesis in the 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 specific and may be general among . Studies on cell fusion between ery- throcyte 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 is injected as a distorted cell into an ultraviolet- irradiated 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 semi- conservative DNA synthesis of polyoma DNA injected into eggs. However, it may not be valid to extend these conclusions to experiments in which large 2+ numbers of Gx nuclei are injected. These nuclei are isolated in -Mg 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 repli- cation is a universal process by injecting nuclei from an invertebrate source, , into Xenopus laevis eggs. Such nuclei do initiate DNA synthesis in response to egg signals. In addition, we show that this DNA syn- thesis, 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 Dx (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 , cornmeal and sugar medium at 25 °C. Eggs were collected for a 1 h 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 (Amer- sham, specific activity 30 Ci/m mole) previously dried under vacuum. For density substitution experiments equal volumes of BrdUTP (20 mM in SUNAP- BSA) 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 autoradio- graphy. 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 per- formed 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/ml final concentration) 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 inverte- brate source, Drosophila melanogaster, would respond to cytoplasmic signals of 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 spon- taneously 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.

Exposure No. of Nuclei scored time eggs r Proportion Nuclei Female no. (days) scored Labelled Unlabelled* labelled Cultured cell 1 7 4 395 1 99-8 2 7 3 248 0 100 3 7 6 185 3 98-4 4* 5 7 291 0 100 Imaginal discs 5 7 8 88 8 91-7 Cultured cell 6 7 6t 0 69 0 * Eggs from female 4 were injected with nuclei, BrdUTP and. [3H]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 con- tained 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 (Table 1) showed that almost every nucleus was autoradiographically labelled. Simi- larly, 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 auto- radiographed. Figure 2 shows the distribution of labelling of the confluent Drosophila cells in one of these experiments. The proportion of cells synthe- sizing 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 20 + Grains per nucleus 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 synthe- sizing 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 40 Fraction number 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 sub- stitution 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 Nuclei % Radioactivity in DNA synthe- time (h) injected HH (1 -770 g/ml) HL (1 -735 g/ml) sized (pg/egg) 3£ None 96 4 13 4 None 95 5 24 94 6 23 5 None 100 0 20 3 Drosophila 59 41 42 45 55 41 5 Drosophila 65 35 99 59 41 46 6 Drosophila 78 22 .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 repli- cation 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 radio- activity 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 under- gone extensive first-round semi-conservative replication. The radioactivity in both the HL and the HH peaks in represents equiva- 13-2 192 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 repli- cation 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 Droso- phila 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 synthe- size 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 ampli- fied 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 autoradio- graphically 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 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 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 iso- lation 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 pro- portion 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 synthe- sized 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 Research Campaign for grants supporting this work.

REFERENCES BENBOW, 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 . Int. Rev. Cytol. 35, 61-116. BROWN, D. D. & LITTNA, E. (1964). Variations in the synthesis of stable RNX's during 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. 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 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 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 (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 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. melano- gaster. 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 tripho- sphate contents of eggs and oocytes of Xenopus laevis. Biochem. J. 127, 597-605.

(Received 23 January 1979, revised 7 September 1979)