DNA synthesis by isolated embryonic nuclei of Xenopus

DEVELOPMENTAL

BIOLOGY

26, 497-502 (1971)

DNA Synthesis

by Isolated

Embryonic

Nuclei of Xenopus

KAREN ARMS Department

of Zoology, Parks Road, Oxford, England’ Accepted July 2, 1971

Nuclei, isolated in sucrose solutions, from adult and embryonic tissues of Xenopus heuis, incorporate TdR-3H when incubated with DNA precursors and tritiated thymidine. This incorporation is DNase-sensitive and requires all 4 deoxyribonucleoside triphosphates. The proportion of nuclei which synthesize DNA in a given time in vitro is very similar to the proportion which do so in uiuo in the tissue or developmental stage from which the nuclei have been isolated. This suggests that nuclei which are in the S phase of the cell cycle at the time of isolation continue to synthesize DNA when incubated in vitro but that DNA synthesis is not initiated or prevented in nuclei by the process of isolating them. INTRODUCTION

The experiments described here were carried out to determine (a) whether nuclei isolated from embryonic tissues synthesize DNA in vitro and (b) whether the pattern of DNA synthesis which they support in vitro is similar to that in uiuo. Nuclei isolated in aqueous media have generally been found to synthesize little DNA (reviewed by Keir, 1965). It seems possible that this is because nuclei have been isolated from tissues in which little DNA synthesis occurs. It was, therefore, of interest to investigate the extent to which nuclei isolated from embryos support DNA synthesis in vitro since, in early embryos at least, DNA is synthesized at a very rapid rate. The whole genome is replicated in as little as 10 min in early cleavage of Xenopus (Graham and Morgan, 1966). DNA synthesis by isolated embryonic nuclei in vitro is here compared with similar synthesis by the same nuclei in uiuo and with DNA synthesis by nuclei from adult frog liver, a tissue in which little DNA synthesis occurs. MATERIALS

AND METHODS

Ovulation and mating of Xenopus laeuis and injection of radioactive precursors 1 Present address: Section of Neurobiology and Behavior, Roberts Hall, Cornell University, Ithaca, New York 14850.

into adults and embryos were carried out by techniques already described (Graham et al., 1966). Nuclear isolation. Nuclei were isolated from the livers of adult and young (up to 8 weeks after metamorphosis) frogs by a method based on those of Gill (1965) and Widnell and Tata (1964). Nuclear isolation was carried out at 4°C throughout. The finely chopped liver was homogenized in 3 times its volume of a solution containing 0.25 M sucrose and 0.003 M MgCl, in a Thomas Teflon-piston homogenizer. The homogenate was then sieved through 8 layers of fine butter muslin and centrifuged for 10 min at 1500 g. The pellet was resuspended in about 0.5 ml of a solution of 0.5 M sucrose-O.001 M MgCl,. This suspension was layered over a solution of 2.4 M sucrose-O.001 M MgCl, and centrifuged for 75 min at 178,000 g. This treatment removes all whole cells and cytoplasmic debris from the resulting pellet, as judged by light microscopy. In the case of adult liver nuclei, some pigment granules are found in the final nuclear pellet. Nuclei were isolated from embryos (stages described by the numerical system of Nieuwkoop and Faber, 1956) as follows. The jelly coats of 1500-2000 embryos were removed chemically by 4 min agitation in a solution of 2@, cysteine-HCl (pH

497

498

DEVELOPMENTAL BIOLOGY

7.8) containing 1% papain. The embryos were then homogenized in 0.1 M sucrose0.003 M MgCl, in a glass homogenizer with a very loosely fitting piston and the homogenate centrifuged for 5 min at 600 g. The pellet was washed 3 times in the homogenizing medium, and the pellet was finally resuspended in 0.5 ml of 0.25 M sucrose-O.001 M MgCl,. This suspension was layered over a discontinuous gradient of 2.4 M, 2.0 M, 1.7 M, 1.2 M, and 0.8 M sucrose containing 0.001 M MgCl, and centrifuged for 75 min at 150,000 g. The nuclei were collected from the interfaces between the 2.4 M and 2.0 M and between the 2.0 M and 1.8 M sucrose layers after this treatment. Estimation of DNA synthesis in vitro. DNA synthesis by isolated nuclei was assayed in a reaction mixture containing, in a final volume of 0.3 ml, 20 fl Tris + HCl (pH 7.8), 7 mM MgCl,, 0.3 fl /3-mercaptoethanol, 0.3 PM each of dCTP, dGTP, dTTP, dATP, and ATP (from Koch-Light Laboratories, Ltd., Colnbrook), 20 &X/ml of tritiated thymidine (TdR-3H, 22.1 Ci/mmole, 1 mCi/ml from The Radiochemical Centre, Amersham), and 0.1 ml of a nuclear preparation, containing about 200 pg of DNA (about 3 x lo7 nuclei). Deoxyribonuclease (DNase) was obtained from British Drug Houses, Ltd. Incubation was carried out at 25°C. Incorporation was usually assayed by precipitating the whole of the incubation mixture onto Whatman GF 81 filters with 5% trichloroacetic acid (TCA) and washing 3 times with 5 ml of 5% TCA before drying the filters for counting in a PPOPOPOP scintillator in a Nuclear Chicago Liquid Scintillation Counter 725. Sometimes, where indicated in the text, DNA was extracted with perchloric acid (PCA) before counting. The nuclear pellet was dissolved in 0.4 N NaOH and the crude nucleic acid precipitated with 0.25 M PCA. Nucleic acid was precipitated by

VOLUME 26, 1971

centrifugation at 40,000 g for 15 min. Solution in NaOH and precipitation with PCA were then repeated twice. DNA was estimated by the Dische diphenylamine reaction as modified by Burton (1956). Nuclear yield was estimated by comparing the DNA content of the filtered homogenate and of the final nuclear pellet. The yield of nuclei from stage 8 embryos was 20-259,; from stage 13 embryos, 55-607;; and from adult liver more.than 90%. Autoradiography. In order to estimate the number of isolated nuclei which had incorporated label during a given time, nuclei were smeared on subbed slides at the end of the incubation period. Such smears were either fixed immediately with methyl alcohol or Perenyi’s fixative or were left unfixed and dried overnight at 35°C. There was no detectable differ-. ence between the results obtained by these two methods and the air-drying procedure was generally adopted. Smears were washed in 10% TCA for at least 20 min and then in running water for 20 min in order to remove any unincorporated label. Embryos for autoradiography were fixed in Perenyi’s fixative and sectioned at 6 p. Slides were dipped in Ilford K2 Nuclear Emulsion, exposed for 14 days at 4OC and processed according to the manufacturer’s instructions. Sections and smears were stained with Mayer’s hemalum and light green before or after autoradiography. RESULTS

Incorporation of Tritiated Thymidine Nuclei Isolated from Gastrulae

by

Figure 1 shows the time course of incorporation of TdR-3H by nuclei isolated from stage 8 embryos (blastulae) and stage 13 embryos (gastrulae). The decrease in total radioactivity recorded in the nuclei after about 70 min of incubation is common in recordings of DNA synthesis by isolated nuclei (e.g., Hyodo and Ono,

ARMS

499

DNA Synthesis in Isolated Xenopus nuclei

age in vivo. About 40% of all nuclei in a stage 8 embryo are synthesizing DNA at any one time. The average cell cycle occupies about 14 min and DNA synthesis is completed in 5-10 min (Graham and TABLE

1

INCORPORATION OF TdR-3H BY NUCLEI ISOLATED FROM STAGE 13-14 EMBRYOS; RESULTS ASSAYED BY Two DIFFERENT M~HODS

Picomoles TdR- 3H incorporated/mg DNA/30 min FIG. 1. The kinetics of TdR-SH incorporation by nuclei isolated from stage 8 (e-0) and from stage 13 (O--O) embryos. The conditions of incubation are described in the text. The embryos from which the nuclei were isolated were from the same mating.

1970). It is possible that the nuclei begin to break down at this time and the DNA they contain is destroyed by nuclease activity. Incorporation is effectively linear for the first 30 min of incubation. Table 1 shows the characteristics of TdR- 3H incorporation by gastrula nuclei in a 30-min incubation under various conditions. The degree to which incorporation of the label was inhibited in the different incubation systems was similar whether incorporation into whole nuclei or into the acid extract was measured. This suggests that incorporation consisted largely of DNA synthesis, not of incorporation of the label into other nuclear components. Comparison of DNA Synthesis by Nuclei from Different Developmental Stages Table 2 shows the extent of TdR-3H incorporation by nuclei isolated from young and adult frog liver, gastrulae and blastulae. There is clearly a considerable difference in the amount of incorporation supported by these different nuclei. DNA synthesis per unit time in vitro was greater, the earlier the developmental stage of the animal from which the nuclei were isolated. This reflects a similar decrease in DNA synthesis with increasing

Incubation

system

Complete systema d?TP, dGTP, dCTP, and dATP omitted Plus DNase at 25 pg/ml

TCA-precipitated whole nuclei

PCA extract

125*

70

(k5)’

tit.5, 29 (57.2) 132 (111)

Plus creatine phosphokmase (0.2 mg) and phosphocreatine (0.4 mM)

a The complete system is described in Materials and Methods. b The blank value of 15 picomoles of TdR-3H/mg DNA per 30 min obtained if boiled nuclei are incubated has been subtracted from all figures. c Figures in parentheses show, as a percentage of incorporation in the complete system, the degree to which the various alterations to the incubation mixture inhibit incorporation. dThe DNase used was from B.D.H. Later experiments show that lower values are obtained if a more highly purified preparation (Worthington) is used. TABLE 2 A COMPARISON OF THE INCORPORATION OF TdR-3H BY NUCLEI

ISOLATED FROM ADULT EMBRYOS”

Source of isolated nuclei

TISSUES AND

Picomoles TdR-3H incorporated mg DNA/30 min Expt. 1

Adult Young Stage Stage

liver liver 12-14 embryos 8-9 embryos

< 10” 35 102 182

Expt. 2 (10 47 139 219

DBlank values, obtained as described in Table 1, have been subtracted from all values given here.

500

DEVELOPMENTAL BIOLOGY

Morgan, 1966). A smaller percentage of the nuclei of a stage 13 embryo are synthesizing DNA at any one time and the S period of endoderm cells occupies about 2 hr. Similarly, cell division occurs at a greater rate in young frog liver, a tissue that is increasing in size, than in adult liver which does not grow (Arms, unpublished). The Proportion of Isolated Nuclei in DNA Synthesis in vitro

Active

Another approach to the problem of how far DNA synthesis in vitro resembles that in vivo, is to determine what proportion of the nuclei of a tissue synthesize

VOLUME 26, 1971

DNA in a given time and to compare this with the proportion of such nuclei that synthesize DNA in the same time after their isolation. The proportion of nuclei synthesizing DNA in a given time will depend upon the relative lengths of the cell cycle and of the S phase. Autoradiography was used to determined the proportion of nuclei, whether in an incubation of isolated nuclei or in an intact tissue, which had synthesized DNA in a given time. Figure 2 shows an autoradiograph of isolated nuclei incubated with TdR-3H and then smeared on a slide. The heavy labeling of some nuclei is in clear contrast to the lack of labeling in others. Most nuclei,

FIG. 2. Autoradiograph of nuclei isolated from stage 13 Xenopus embryos and incubated with TdR-3H for 60 min (see Materials and Methods for experimental details). The photograph was taken in the plane of the autoradiographic grains. For this reason, unlabeled nuclei are not very clearly defined.

ARMS

TABLE PROPORTIONS OF NUCLEI

SOURCES WHICH ARE LABELED in Viva AivD AFTER ISOLATIOP

Stage 8 embryos 60

Stage 13 embryos 60

labeledb labeledb

2044 9 1991 97.84:;

labeled* labeledb

400’ 1 393 98.50%

Length of incubation Isolated nuclei Number counted Number weakly Number heavily Total labeled Nuclei in uiuo Number counted Number weakly Number heavily Total labeled

3

FROM VARIOUS

Source of nuclei: (min):

501

DNA Synthesis in Isolated Xenopus nuclei

AFTER EXPOSURE TO TdR-3H

Yowrfrog

Adult liver

60

60

1950 521 619 58.464;

2003 16 130 7.28%

210 l? 0 0.1r;

730’ 156 304 63.01c;

5006 17 35 10.4oc,

500d 1 1 0.4c;

a Embryos were injected with about 100 rnpl of an aqueous solution of TdR-3H to give a final concentration of label inside the embryo of about 1 &i/ml. Embryos were fixed 30 or 60 min after injection. Figures for 60 min exposure to label are given. Figures for 30 min exposure were also obtained. Frogs were injected intraperitoneally with 0.5 PCi TdR-3H per gram body weight. bWeakly labeled: labeled up to twice background level (background = less than 4 grains per nucleus). Heavily labeled: labeled more than twice background. c Nuclei counted were from all parts of 5 embryos obtained from 2 different matings. d Figures for adult liver nuclei in viuo are for a 2-hr exposure to label. The figures for young liver are for 90 min labeling.

therefore, incorporate the label extensively or not at all in a period of 60 min. Table 3 shows that the proportion of nuclei which synthesize DNA in a given period in vitro is, in general, very similar to that in the tissue of origin. The fact that the proportions of nuclei which synthesize DNA in a 60-min period in vivo and in vitro are so similar strongly suggests that most of those nuclei which are synthesizing DNA in vivo at any one time, continue to do so under the conditions of isolation and incubation in vitro described here. DISCUSSION

The evidence presented here demonstrates that nuclei isolated from frog embryos incorporate tritiated thymidine into an acid-insoluble form in vitro. The fact that incorporation is largely dependent upon the presence of all 4 deoxyribonucleoside triphosphates and that most of the label is extractable into a semipurified

DNA fraction, strongly suggests that incorporation is, at least mainly, into DNA under the influence of a replicative transferase. This is further suggested by the fact that incorporation is strongly inhibited by DNase (Table 1). The results suggest that those nuclei which are synthesizing DNA at any given time in vivo continue to do so after isolation. The evidence for this is 2-fold. First, nuclei isolated from animals of younger developmental stages, which are those most active in cell division and DNA synthesis in vivo, are those which incorporate most tritiated thymidine in a given time after isolation. Similar results of other workers have shown that nuclei isolated from adult tissues synthesize relatively little DNA (Keir, 1965). Loeb et al. (1967) have shown that nuclei from cleavage stages of sea urchins synthesize a considerable quantity of DNA in vitro, and Brewer and Rusch (1965) have shown that DNA synthesis by isolated nuclei of Physarum

502

DEVELOPMENTAL BIOLOGY

occurs only in those nuclei isolated during the DNA-synthesizing part of the cell cycle. Second, the fact that the proportion of nuclei that incorporate TdR-3H in a given time in vitro closely resembles that in the tissue from which the nuclei have been isolated, suggests that nuclei synthesizing DNA in uiuo, and no others, continue to do so after isolation. If nuclei are not induced by isolation to start or to cease DNA synthesis, as the evidence presented here suggests, many questions still remain as to how far isolation alters the normal cell cycle. For instance, nuclei from stage 8 embryos would be expected to pass through the entire cell cycle up to 3 times during the period of 60 min for which they are incubated in vitro. It might be expected that entry into mitosis would be represented by nuclear breakdown in vitro but, in fact, many stage 8 nuclei are intact after 60 min incubation. Does this mean that, in uitro, these nuclei become blocked in the S phase of the cell cycle or in G2 and never enter mitosis? The answer to this question is not known. From the fact that the proportion of nuclei which become labeled with TdR-3H in vitro increases little, if at all, between 30 and 60 min after the start of an incubation, it appears that nuclei do not enter the S phase in vitro, but the evidence presented here does not show whether or not isolated nuclei may stop DNA synthesis: that is, leave the S phase. The simplest explanation of the results polycephalum

VOLUME 26, 1971

obtained, would be that nuclei remain, after isolation, in that phase of the cell cycle in which they were isolated. Most of the work described here was performed during the tenure of a Research Scholarship from the Science Research Council. I am most grateful to Dr. J. B. Gurdon for helpful comments on the manuscript. REFERENCES

BREWER,E. N., and RUSCH, H. P. (1965). DNA synthesis by isolated nuclei of Physarum polycephalum. Biochem. Biophys. Res. Commun. 21, 235241. BURTON, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. GILL, D. M. (1965). An improved method for the isolation of rat liver nuclei by density centrimgation. J. Cell Biol. 24, X7-161. GRAHAM, C. F., and MORGAN, R. W. (1966). Changes in the cell cycle during early amphibian development. Develop. Biol. 14, 439-460. K., and GURDON, J. B. (1966). GRAHAM, C. F., ARMS, The induction of DNA synthesis by frog egg cytoplasm. Develop. Biol. 14, 349-381. HYODO, M., and ONO, T. (1970). Regulation of nuclear DNA synthesis in rat liver. Exp. Cell Res. 60, 401-404. ’ KEIR, H. M. (1965). DNA polymerases from mammalian cells. Prog. Nucl. Acid Res. Mol. Biol. 4, 81-128. LOEB, L. A., MAZIA, D., and RUBY, A. D. (1967). Priming of DNA polymerase in nuclei of sea urchins by native DNA. Proc. Nat. Acad. Sci. U.S. 57, 841-848. NIEUWKOOP, P. D., and FABER, J. (1956). “Normal Table of Xenopus laeuis (Daudin).” North-Holland, Amsterdam. WIDNELL, C. C., and TATA, J. R. (1964). A procedure for the isolation of enzymically active rat-liver nuclei. Biochem. J. 92, 313-317.