Incorporation of deoxythymidine triphosphate into nuclear DNA of developing frog embryos in vitro

DEVELOPMENTAL

BIOLOGY

Incorporation

29,

214-219

(19%)

of Deoxythymidine Triphosphate into Nuclear Developing Frog Embryos in Vitro

DNA of

J. KLOSE AND R. A. FLICKINGER Department

of Biology,

State

of New York, Buffalo, New York 14214

University

Accepted

March

30, 1972

The rate of incorporation of labeled precursor into DNA of isolated nuclei decreased from gastrulation to the tailbud stage in developing frog embryos.The addition of saturating amounts of calf thymus DNA revealed that the activities of the enzyme(s) which incorporates deoxythymidine triphosphate (dTTP) into DNA in vitro decreased over this period. Native DNA was a better template than denatured DNA. The addition of a purified microbial DNA polymerase to gastruia and tailbud nuclei showed that the chromatin DNA of the later stage is a poorer template for the enzyme. The activity of the enzyme(s) which accounts for dTTP incorporation into DNA in vitro was higher during the early S period of neurulae than for late S and higher for late S than early S of tailbuds when nuclei were isolated from partially synchronized cells. For both the randomly dividing and partially synchronized cells, the activity of the enzyme(s) incorporating dTTP into DNA in vitro is correlated with the level of incorporation of labeled precursor into DNA in uiuo.

way, 1940), neurulae (stage 14), and tailbuds (stage 18) at 4°C. Embryos were The rate of DNA synthesis in developwashed with 0.7% KC1 solution and then ing amphibian embryos decreases as cell with solution A (0.012 M Tris. HCl, pH determination and differentiation occur (Gurdon, 1968). Furthermore, a greater 7.8, 0.006 M KCI, 0.003 M MgC12, 0.25 M sucrose) and homogenized in solution A portion of the DNA becomes late-replicating during this period (Stambrook and using a Teflon-glass homogenizer. The homogenate was poured through four layers Flickinger, 1970; Remington and Flickinger, 1971). The purpose of the present of cheesecloth and centrifuged at 600 g study was to determine the activity of the for 10 min. The pellet was resuspended in 2.2 M sucrose (containing 0.001 M MgCl,) which incorporates dTTP enzyme(s) was into DNA in vitro, as well as the endoge- by homogenization. Centrifugation nous level of dTTP incorporation into performed in a swinging-bucket rotor DNA in nuclei isolated from developing 25.1 at 50,000 g for 1 hr. The nuclear frog embryos (Ranu pipiens). Enzyme ac- pellet was washed with solution A by tivity also was determined during early centrifugation at 600 g for 10 min. Aliquots of the nuclear preparation conand late S periods for frog embryo explants which had been partially synchronized in taining about 400 pg DNA were resustheir cell cycles in order to correlate the pended in 1.5 ml solution A and sonicated activity of the enzyme with the rate of with the microtip nine times for 5-set synthesis of the DNA in the early and late periods (Sonifier Cell Disruptor, Model W 140 D). The homogenate was then adS period in uivo. justed with solution A to a concentration METHODS of 30 pg DNA/O.2 ml. Isolation of nuclei. Nuclei were isolated Enzyme assay. Assays for the incorporafrom dejellied gastrulae (stage 10, Shumtion of dTTP into DNA were carried out *This research was supported by grants from the with a reaction mixture containing the National Institutes of Health (GM. 16236-02) and the National Science Foundation (GB 8029). following components in a final volume of INTRODUCTION

214 CopyrIght All rights

0 1972 by Academic Press. Inc. of reproduction in any form reserved

KLOSE

AND

FLICKINGER

D-TTP

0.5 ml; 40 pmoles Tris.HCl (pH 78, 0.1 pmole each of dATP, dGTP, dCTP, 0.8 nmole dTTP, 2.5 &i H3-dTTP (specific activity 11.1 Ci/mmole), 4 pmoles 1 pmole KCl, 0.5 @mole sodium M&L, versenate, 0.5 pmole mercaptoethanol, and a sonicated nuclear preparation containing 30 pg DNA. In experiments in which the activity of the enzyme(s) involved in dTTP incorporation was assayed, 100 pg of native or denatured calf thymus DNA (Worthington Biochemical Corp) was also added. Calf thymus DNA, dissolved in 0.01 A4 Tris.HCl, pH 7.8 (2 mg DNA/ml), was denatured by heating at 100°C for 15 min and then cooling immediately at 0°C. Endogenous activity of nuclear preparations was determined in the same reaction mixture with the omission of calf thymus DNA. All glassware was heat-sterilized and all stock solutions were made with sterile double-distilled water containing penicillin (50 units/ml) and streptomycin sulfate (50 pg/ml). The reaction mixture was prepared at O”C, then incubated at 30°C for 15 min; and the reaction was terminated with 2 ml of cold 10% TCA containing 1% sodium pyrophosphate. The precipitate was washed 4 times with 5% TCA-1% sodium pyrophosphate by centrifugation and collected on 4 glass filters (934 AH Glass Fiber Filter, Reeve Angel). The precipitates then were washed twice more on the filters with the same solution. The filters were dried with an infrared lamp and counted in a liquid scintillation counter in a toluene-PPO-POPOP solution. Cell synchronization. Explants of neurulae and tailbuds were partially syn5-fluorodeoxyuridine chronized using (FdUrd) as described by Remington and Flickinger (1971). Nuclei were isolated from cells 2 hr (early S phase) and 6 hours (late S phase) after release of the block of DNA synthesis. The duration of the S period in (FdUrd)-treated cells in 8-9 hours (Remington and Flickington, 1971).

Incorporation

into

DNA

215

RESULTS

Characteristics of the Assay System Experiments performed under different conditions show the ability of sonicated nuclei of frog embryos containing 30 gg of frog DNA to catalyze the incorporation of labeled deoxythymidine triphosphate into acid insoluble material. While a relatively low activity is found in the endogenous system, the incorporation of the labeled precursor can be increased by adding high molecular weight calf thymus DNA (Worthington Biochemical Corp.) (Fig. 1). Native calf thymus DNA is a better template than denatured DNA. When increasing amounts of native DNA were added to the reaction mixture, the activity increased and the saturation level was achieved at 100 jog DNA of added calf thymus DNA for all three embryonic stages (Fig. 2). Control experiments were performed with the standard reaction mixture lacking the nuclear preparation and also with the complete reaction mixture which was precipitated with TCA at zero time. In both cases the radioactivity found in the precipitate did not exceed the background of the liquid scintillation spectrometer. When dATP, dGTP, and dCTP were omitted from a reaction mixture containing tailbud nuclei and native calf thymus DNA, the level of H”-dTTP incorporation decreased by 85% In order to determine if there was DNase activity in the nuclear preparations during the incubation period of 15 min, 1.6 pg of labeled frog DNA (2000 cpm) was added to 0.5 ml of the reaction mixture containing 0.2 ml of the tailbud nuclear preparation. Incubation and termination of the reaction was carried out as described and after centrifugation aliquots of the supernatant were counted. The radioactivity did not exceed that measured in samples precipitated at zero time. Control experiments with 1 kg of electrophoretically pure DNase 1 (Worthington Biochemical

216

DEVELOPMENTAL

BIOLOGY

VOLUME

29, 1972

37°C with 500 pg electrophoretically pure DNase 1 (Worthington Biochemical Corp.) in 1.0 ml of 0.01 M Tris .HCl, pH 7.5, 2 mM MgCl, converted all this radioactivity to an acid-soluble form. Enzyme Activity Stages

30 GASTRULA LsT.10)

I 40

I 50

I I 60 70 NEURULA (ST. 141

I 60

1 90 too TAIUUD (ST.16)

HOURS

Frc. 1. Incorporation of dTTP into DNA of sonicated nuclei of developing gastrulae (stage lo), neurulae (stage 14), and tailbuds (stage 18). (Experiments were performed two times in triplicate.) The horizontal axis (hours) refers to hours after fertilization at 18°C. The systems contained sonicated nuclear preparations of each stage containing 30 pg frog nuclear DNA, 40 rmoles Tris.HCl (pH 7.8) 0.1 pmoles each of dATP, dGTP, dCTP, 0.8 nmoles dTTP, 2.5 PCi H3-dTTP, 4 rmoles MgCl,, 1 pmole KCl, 0.5 pmole sodium Versenate and 0.5 pmoles mercaptoethanol in a volume of 0.5 ml. Native and denatured calf thymus DNA (100 /rg DNA) were added in two of the series of experiments. Legends: 0 for endogenous system, no calf thymus DNA added; x for 100 pg denatured calf thymus DNA added; 0 for 100 fig native calf thymus DNA added.

Corp.) per 0.5 ml of mixture show that 35% of the radioactive DNA was rendered acid soluble after a 15-min incubation. It appears that there is no detectable DNase activity in tailbud nuclei under the conditions of our experiments. To ascertain that the labeled dTTP actually entered DNA in our experiments, DNA was isolated according to Kohl et al. (1969) from an enzyme reaction mixture containing tailbud nuclei and calf thymus DNA. Counting revealed this DNA to be radioactive, and incubation for 8 hr at

at Different

Embryonic

As shown in Fig. 1, the activity of the enzyme(s) incorporating dTTP into DNA in vitro decreases from the gastrula to the tailbud stage. This is true of the endogenous system, as well as the exogenous system containing native or denatured calf thymus DNA. The preference for native DNA, as compared to denatured DNA, is greater for the enzyme(s) of tailbud nuclei than for gastrula nuclei. Experiments were performed in which equal amounts of nuclear preparations from gastrula and tailbuds were used in the reaction mixture separately or together. The experiment done with the mixture revealed the summation of activities of the single experiments (Table 1). In order to obtain some information about the template activity of DNA at different developmental stages, saturating

.

GQSTRULA

.

FIG. 2. Effect of addition of different amounts of native calf thymus DNA (horizontal axis) on incorporation of dTTP into DNA of sonicated nuclei of developing frog embryos containing 30 rg DNA The constituents of the system are similar to those described in the legend for Fig. 1. Legend: 0 for stage 10 gastrula nuclei; x for stage 14 neurula nuclei and 0 for stage 18 tailbud nuclei.

KLOSE

~~~~~~~~~~~~~~~~~ INTO DNA OF

Source

AND FLICKINCER

D-TTP

TABLE 1 TRIPHOSPHATE INCORPORATION A MIXTURE OF GASTR~JLA AND TAILBCD NUCLEIC of nuclei

Endogenous system fimles dTTP incorporated/ minl30 pg frog nuclear DNA i SD

Gastrulae

(stage

101, 30 pg DNA

Tailbuds

(stage

18), 30 wg DNA

Gastrula-tailbud each of DNA ‘The (pH 7.8), unlabeled 4 rmoles Versenate, cated frog of 0.5 ml.

mixture,

30 pg

40.7 12.6 10.7 S1.2 54.3 +6.8

x x X X x x

10-s 10-g 10-g 10-g 10-S 1O-9

systems contained 40 bmoles Tris. HCl WdTTP (2.5 &i), 0.1 pmole each of dATP, dGTP, dCTP, 0.8 nmole dTTP, MgCl,, 1 pmole KCI, 0.5 rmole sodium 0.5 rmole mercaptoethanol and soninuclei containing 30 pg DNA in a volume Experiments were performed four times.

amounts of bacterial DNA polymerase (M. lysodeikticus, 15.55 units; spec. act. 19.8 units/mg protein; Miles Laboratories, Inc.) were added to the standard reaction mixture containing the nuclear preparations (DNA content 30 pg) from gastrulae or tailbuds. The incorporation of labeled deoxythymidine triphosphate was 23% higher in the presence of gastrula nuclei than tailbud nuclei (Table 2). Enzyme Activity in the Early and Late SPeriod of Neurula and Tailbud Cells The activity of the nuclear enzyme(s) incorporating dTTP into DNA irl vitro was considerably higher (up to 3-fold greater) when assayed during early or late S period of partially synchronized cells than that of nuclei from randomly growing cells (Fig. 1, Table 3). The enzyme activity of neurulae is higher in early S than in late S. In contrast, tailbuds reveal a higher activity in the late S period (Table 3). DISCUSSION

Loeb concludes from his investigations of sea urchin embryos (Loeb et al., 1967) that DNA polymerase requiring double-

Incorporation

into

217

DNA

stranded DNA becomes prominent in eukaryotic cells only where there is active reproduction of genetic material, as in developing embryos or in regenerating rat liver (Mantsavinos and Munson, 1966). Therefore the preference for native DNA of frog embryo nuclei in our in vitro system may indicate the presence of a replicative polymerase. Furthermore, the correlation between the activity of the enzyme(s) promoting the incorporation of dTTP into acid-insoluble material in vitro and the rate of DNA synthesis in uivo suggests the presence of a replicative enzyme. The enzyme activity remaining after 3 deoxynucleotide triphosphates are omitted can probably be attributed to the presence of natural DNA precursors in the sonicated nuclei. In other reports this incorporation of precursor into DNA when one of the 4 deoxynucleoside triphosphates is omitted is attributed in part to a terminal DNA polymerase (Keir, 1965). But the presence of a repair enzyme can also account for this finding. The results of this investigation reveal a decrease in the endogenous level of incorporation of labeled precursor into DNA of sonicated nuclei as determination and differentiation occur in developing frog embryos (Fig. 1). The addition of saturating amounts of calf thymus DNA to the reaction mixture shows that the activity of the enzyme(s) incorporating dTTP into DNA in vitro decreases over TABLE 2 TEMPLATE ACTIVITY OF GASTRULA AND TAILBUD NUCLEI IN THE PRESENCE OF MICROBIAL DNA POLYMERASE” Source of nuclei (30 pg DNA) Gastrula (stage Tailbud (stage

@Moles dTTP incorporated/ min/30 fig frog nuclear DNA zt SD 905.4

x 10-v

* 6.4

x 10 9

704.9

x 10-9

* 17.1

X 10-g

10) 18)

--..-o DNA polymerase (15.5 units) of Micrococcus lysodeikticus was added to the reaction system described in the legend of Table 1. Experiments were performed eight times.

218

DEVELOPMENTAL

dTTP

INCORPORATION

Hours after release fluorodeoxyuridine inhibition

of

INTO DNA

BIOLOGY

TABLE OF SONICATED

PMoles

dTTP Exogenous

VOLUME 3 NUCLEI

29, 1972

DURING

incorporated/min/30 system,

EARLY

AND

LATE

c(g frog nuclear

100 pg native

calf thymus

Neurula nuclei, 30 pg DNA 2 Hr (early S period) 6 Hr (late S period) “The reaction added. Experiments

215.9 186.7

x 1O-9 z+z 6.3 x 1O-9 zt 4.0

DNA

+ SD

DNA

Tailbud nuclei, 30 pg DNA x 1O-9 x 10m9

system is described in the legend of Table were performed six times in duplicate.

the same period (Fig. 1). A decrease of DNA polymerase activity occurs in developing sea urchin embryos during gastrulation and is correlated with a reduction in the rate of cell division and DNA synthesis (Mazia, 1963; Fansler and Loeb, 1969). This same relation apparently may hold for the period of cell determination and differentiation of developing amphibian embryos since the rate of cell division and DNA synthesis decreases (Flickinger et al., 1967; Gurdon, 1968) over the period when enzyme activity is reduced (Fig. 1). It is unlikely that the presence of considerable DNase activity at later stages accounts for the reduced incorporation of labeled precursor into DNA in vitro since no DNase activity was detected in tailbud nuclei. However, it is possible that a nicking enzyme (Burgoyne et al., 1970), whose activity is so low we cannot detect it, may contribute to the observed results. Native DNA is the preferred template for the DNA polymerase of developing sea urchin embryos (Loeb et al., 1967), and this is also true of the frog embryo enzyme(s) (Fig. 1). The preference for native DNA is more prominent at later stages of development. Denatured DNA may be a poorer template for the same enzyme or it is possible that there are two kinds of enzyme preferring the native and denatured DNA, respectively (Chiu and Sung, 1970). The reduction of DNA synthesis at later stages could be due to an inhibitor of DNA synthesis that accumulates in cells

S PERIODS

1. Native

195.5 276.3 calf

1o-9 l 14.4 x 10-s x 1O-9 i 7.9 x 1O-9

x

thymus

DNA

100 pg, was also

(Fansler and Loeb, 1967; Chiu and Sung, 1970). However, in the mixture of sonicated nuclei the tailbud nuclei (low DNA enzyme activity) did not inhibit the activity of gastrula nuclei (high activity). Furthermore, the gastrula nuclei did not stimulate the activity of the tailbud nuclei (Table 1); the results were additive. This experiment does not support the idea that there is an inhibitor located in nuclei which accounts for the reduced enzyme activity at later stages, but such an inhibitor could have a cytoplasmic localization. The addition of excess DNA polymerase of Micrococcus lysodeikticus to sonicated gastrula and tailbud nuclei reveals that the template activity of the tailbud chromatin is less than that of the gastrula chromatin (Table 2). However, this decrease in template activity (Table 2) is much less than the decrease in endogenous activity of H3-dTTP incorporation into DNA between the gastrula and tailbud stages (Fig. 1). The activity of the enzyme(s) incorporating dTTP into DNA in uitro, on the contrary, shows a marked reduction over this period of development (Fig. l), and it appears that this change may play a large role in affecting DNA synthesis. Although the microbial DNA polymerase may be primarily a repair enzyme (deLucia and Cairns, 1969), if the repair function is related t,o replication our results may reflect the template efficiencies of the two nuclear preparations. There are two waves of DNA synthesis during the S period which correspond to

KLOSE

AND FLICKINCER

D-TTP

early and late replicating DNA in partially synchronized frog neurula and tailbud cells (Remington and Flickinger, 1971). At the neurula stage the major portion of the DNA is early replicating (2 hr after start of S), whereas at the tailbud stage the greater part of the DNA is late replicating (6 hr after start of S). At the same two points of time in the cell cycle (2 hr and 6 hr) the activity of the dTTP incorporation enzyme(s) was determined. The results show a higher activity in early S than for late S of neurulae and a higher enzyme activity for late S than for early S of tailbuds. At both stages the higher enzyme activity is associated with the DNA that is replicating more rapidly in uiuo. Mouse heterochromatin is known to be late replicating (Tobia et al., 1970), and it has been determined that the isolated heterochromatin fraction of mouse liver had a more rapid rate of incorporation of labeled DNA precursor in uitro, as well as a higher DNA polymerase activity, than does the euchromatin fraction (Klose and Flickinger, 1971). It is possible that the rapidity of replication of late-replicating DNA (Schmid and Leppert, 1969; Comings, 1970; Wright et al., 1970) may cause the restriction of transcription which is usually observed in heterochromatin. This inverse relation between rate of DNA replication and transcription is also evident during cleavage of the amphibian embryo (Gurdon, 1968) and may possibly be a fundamental control mechanism that regulates transcription. REFERENCES BURGOYNE, L. A., WAGAR, M. A., and ATKINSON, M. R. (1970). Calcium-dependent priming of DNA synthesis in isolated rat liver nuclei. Biochem. Biophys. Res. Commun. 39,254-259. COMINGS, D. E. (1970). Quantitative autoradiography of heterochromatin replication in Microtus agrestis. Chromosoma 29,434-445. CHIU, J.-F., and SUNG, S. C. (1970). DNA nucleotidyltransferase activity of the developing rat brain. Biochem. Biophys. Acta 209, 34-42. DELUCIA, P., and CAIRNS, J. (1969). Isolation of an

Incorporation

into

DNA

219

E. coli strain with a mutation affecting DNA polymerase. Nature (London) 224, 1164-1166. FANSLER, B., and LOEB, L. A. (1969). Sea urchin nuclear DNA polymerase. II. Changing localization during early development. Exp. Cell Res. 57, 305310. FLICKINGER, R. A., FREEDMAN, M. L., and STAMBROOK, P. J. (1967). Generation times and DNA replication patterns of cells of developing frog embryos. Deuetop. Biol. 16,457-473. GURDON, J. (1968). Nucleic acid synthesis in embryos and its bearing on cell differentiation. Essays Biothem. 4,26-68. KEIR, H. M. (1965). Progr. Nucleic Acid Res. Mol. Biol. 4, 81-128. KOHL, D. M., GREENE, R. F., and FLICK~GER, R. A. (1969). The role of RNA poiymerase in the control of RNA synthesis in vitro from Rana pipiens embryo chromatin. Biochim. Biophys. Acta 179, Z-38. KLOSE, J., and FLICKINGER, R. A. (1971). Exp. Cell Res. LOEB, L. A., MAZIA, D., and RUBY, A. D. (1967). Priming of DNA polymerase in nuclei of sea urchin embryos by native DNA. Proc. Nat. Acad Sci. U.S. 57, 841-848. MAZIA, D. (1963). Synthetic activities leading to mitosis. J. Cell Comp. Physiol. 62, Suppl. 1, 123140. MANTSAVINOS, R., and MUNSOE;, B. (1966). Studies on the synthesis of deoxyribonucleic acid by mammalian enzymes. II. An investigation of the primer requirement of partially purified regenerating rat liver deoxyribonucleic acid nucleotidyltransferase. J. Biol. Chem. 241,2840-2844. REMINGTON, J. A., and FLICKINGER, R. A. (1971). The time of DNA replication in the cell cycle in relation to RNA synthesis in frog embryos. J. Cell Physiol. 77, 411-422. SCHMID, W., and LEPPERT, M. F. (1969). Rates of DNA synthesis in heterochromatic and euchromatic segments of the chromosome complements of two rodents. Cytogenetics 8,125-135. SHUMWAY, W. (1940). Stages in the normal development of Rana pipiens. Anat. Rec. 78, 139-148. STAMBROOK, P. J., and FLICKINGER, R. A. (1970). Changes in chromosomal DNA replication patterns in developing frog embryos. J. Exp. Zool. 174, lOl114. TOBIA, A. M., SCHILDKRAUT, C. L., and MAIO, J. J. (1970). Deoxyribonucleic-acid replication in synchronized cultured mammalian cells. I. Time of synthesis of molecules of different average guanine + cytosine content. J. Mol. Biol. 54,499-515. WRIGHT, W. C., MUKHERJEE, B. B., MANN, K. E., GHOSAL, S. K., and BURKHOLDER, G. D. (1970). Quantitative autoradiographic analysis of rates of DNA synthesis in X-chromosomes of bovine females. Exp. Cell Res. 63, 138-142.