The synthesis of histone H1 during early amphibian development

DEVELOPMENTAL

BIOLOGY

The Synthesis

75, 222-230(1980)

of Histone Hl during Early Amphibian J.M.

Development

FLYNN AND H.R. WOODLAND

Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, England Received January 2, 1979; accepted in revised form May 29, 1979 There are at least three Hl histones in Xenopus. These change in synthetic rates during development, but there is no evidence for stage-specific histones. HI is made at a low rate in oogenesis, but since the other histones are also made at low rates, the relative synthetic rate of HI is comparable to the rates at which each of the other histones is made. The non-HI histones show a large, abrupt increase in their synthetic rate during oocyte maturation (Adamson, E. D., and Woodland, H. R. (1977). Develop. Biol. 57, 136-149), but the rate of HI synthesis remains relatively low into cleavage. It increases during cleavage, until HI and other histones again have comparable rates of synthesis by the late blastula stage. Evidence is presented that the low rate of HI synthesis measured in eggs and cleaving embryos is not an artifact produced by poor recovery of HI from these developmental stages. Previous work has shown that at these early stages the synthesis of all histones is under posttranscriptional control. The mechanisms responsible must therefore be wholly or partly separate for Hl and the other histones. INTRODUCTION

We have previously described in some detail the developmental program by which Xenopus embryos make histones other than Hl (Adamson and Woodland, 1974, 1977; Woodland and Adamson, 1977). The non-H1 histones are synthesized at a steady low rate during oogenesis, so that the egg contains a substantial pool for use later in development. When steroid hormones induce the oocyte to become an egg (maturation), the rate of non-H1 histone synthesis rises 20- to 50-fold, becoming much higher than is needed for the immediate construction of new chromatin. This increase occurs at the time that the oocyte nucleus breaks down, through a posttranscriptional process. The oogenetic store of histones is thus further supplemented in the early stages of cleavage. Even so, by the early gastrula stage the non-H1 histone pool, accumulated in oogenesis, should have been exhausted, according to our previous calculations (Woodland and Adamson, 1977). Adamson and Woodland (1974) reported

that Hl histones are made in scarcely detectable amounts in eggs, and only at low rates during cleavage. Not until the late blastula stage were large amounts of Hl synthesis detected. It was suggested that these results could have three explanations: All of the Hl could be stored in oogenesis, some unusual form of Hl could be made in cleavage, and cleavage nuclei could be deficient in Hl. To these could be added the possibility that there was a loss of Hl during extraction from early-stage embryos. In the work reported here we have tried to find out whether oocytes store Hl, to confirm that Hl synthesis is really deficient at early stages, and to measure rates of Hl synthesis through early development. These changes in synthetic rate are of special interest in relation to posttranscriptional control, since unfertilized eggs and blastulae contain comparable amounts of Hl mRNA (Ruderman et al., 1979). MATERIALS

Oocytes, eggs, and embryos of Xenopus laeuis were obtained, labeled with isotopic 222

0012-1606/80/030222-09$02.00/O Copyright All rights

0 1980 by Academic Press, of reproduction in any form

Inc. reserved.

AND METHODS

FLYNNANDWOODLAND

Histone Hl Synthesis in Early

precursor, and otherwise handled as described previously (Adamson and Woodland, 1974, 1977; Woodland and Adamson, 1977). Marker Xenopus erythrocyte histones were made by the method described in the first of these papers. Embryos were staged according to Nieuwkoop and Faber (1956). The guanidinium chloride/ethanol extraction medium (GuCl/EtOH) used in previous studies (10% guanidinium chloride and 75% ethanol) was modified, so that Hl histone was extracted, by reducing the ethanol concentration to 40% (v/v). Otherwise the extraction and electrophoresis were conducted as described by Woodland and Adamson (1977), except that after centrifugation of the initial homogenate for 10 min at lO,OOOg,histones were precipitated with 6 vol of ethanol at -20°C overnight. This gave lower contamination by nonprotein material than acetone precipitation. ““I-Histones were prepared as described by Adamson and Woodland (1974), except that the iodination mixture contained 4 M urea. In most experiments calf thymus histone was used, although we also used perchloric acid-purified Xenopus erythrocyte histone. The iodinated histone was diluted with nonradioactive histone to give a final concentration of 2 mg/ml. This was to inhibit the loss of histone by binding to almost any available surface. This can be a serious problem when trace amounts of histone are handled. “‘1 Radioactivity was determined in an LKB/Wallac y-counter. Tritium radioactivity in gel fragments was measured after incubating the dried gel fragments for several days in scintillation fluid containing 4 g PPO, 0.1 g POPOP, 10 ml water, 90 ml Soluene (Packard), and toluene to 1 liter. The determination of total and acid-insoluble radioactivity was as described by Adamson and Woodland (1974). Rates of histone synthesis were calculated from 2D histone spots using the formula:

Decbelopment

223

pg made per hr (cpm in histone) X (mw of histone) X (pmoles lysine in pool) X 100 ~~__~(hr of incorporation) x (cpm in soluble pool) x (lysine residues per histone) X (5%recovery). It is assumed that incorporation is linear, which is essentially true over the long incubation times used, and when the precursor used is lysine, since lysine has a large pool in all developmental stages. The Xenopus lysine pool is smaller than that of Rana pipiens oocytes, and lysine labeling does not give the artifacts found in Rana by Shih et al. (1978). For a given developmental stage the formula can be simplified to pg made per hr = (proportion of soluble cpm incorporated per hr into histone) x constant. The value of the pool for oocytes was taken to be 150 pmole and for eggs 200 pmole. The lysine pool remains constant in size through early development, so the egg value was used for embryos from cleavage to the gastrula stage (Adamson and Woodland, 1977). The recovery values used were those shown in Table 2. Total histone synthetic rates were calculated from the rates determined for each individual histone. The use of our previously determined values for pool size can be justified by the fact that we obtain values for non-H1 histone synthesis that are similar to those found in our previous studies. In the latter, radioactive incorporation and amino acid pool sizes were determined on cells from the same ovary or mating. RESULTS

Extraction of Hl Histones from Embryos at Different Developmental Stages In previous studies we have used a number of solvents to extract histones from

H3 HZ H4

FIG. 1. 2D electrophoretic patterns of Xenopus histories. The first dimension (left to right) was 2.5 M urea/ 0.9 M acetic acid in A and C, and 6.25 M urea/O.9 M acetic acid in the remaining figures. The second dimension was 18% acrylamide/SDS. In B and D-G the gels are fluorographs of proteins labeled with [JH]lysine in uiuo, and the hand-drawn circles mark the positions of erythrocyte histones analyzed on the same gel. (A) Erythrocyte histones stained with Coomassie brilliant blue. On 2.5 M urea gels the H2A runs faster, in the first dimension, relative to the other histones, than on 6.25 M urea gels. (B) Gastrula histones synthesized from stages 10 to 16. (C) An enlargement of the erythrocyte HI region seen in A. (D) An enlargement of the Hl region of a fluorograph of newly synthesized histones made in the stage 6 oocyte. (E) An enlargement of the HI region of Hl made in the unfertilized egg. A cleaving embryo gives an identical picture. In this and in F and G the identity of the histones is the same as in D. (F) The newly synthesized HI of the blastula, made from stages 8 to 9. (G) An enlargement of the HI region of B. 224

FI,YNN AND WOODLAND

Histone HI Synthesis in Early l)evelopment

whole embryos of Xenopus (Adamson and Woodland, 1974). We routinely used 75% ethanol/lo% guanidinium chloride, which recovers only classes of histone other than Hl. We also used 5% perchloric acid, which extracts only the Hl histones, and 0.4 N HzS04, which recovers all of the histones. The acid solvents extract much nonprotein material, such as carbohydrate, which interferes with subsequent gel electrophoresis. In the work described here we have used 40% ethanol/lo% guanidinium chloride, which extracts Hl, as well as the other main histone classes. For the latter the results are similar to those obtained by Adamson and Woodland (1974), except that H4 recovery is sometimes poor. This solvent more rarely may give a low recovery of Hl. This is detected by examining the stained marked Hl proteins on the 2D gel, and the experiment may then be ignored. The 2D gel electrophoretic patterns of newly synthesized histones are shown in Fig. 1. They are also compared to the Hl histones of the adult erythrocyte, initially analyzed in this way by Adamson and Woodland (1974). In the erythrocyte histone three Hl species are resolved. These we call Hl*, HUB, and Hlc. Cassidy and Blackler (1978) previously noted two species of HI in Xenopus laevis, which they

~~. ~-

Expt 1 Injected Injected jetted Expt 2 Injected Injected jetted

225

called Hl’ and Hl”. However, they called the Hl of Xenopus borealis Hl” and Hl”‘, so to avoid confusion we have introduced different nomenclature. Two species of Xenopus laevis Hl have also been reported by Destree et al. (1972, 1973), using acid/ urea gel electrophoresis. Two-dimensional gels are needed to resolve fractions HUH and Hlc by electrophoresis. Three Hl histones were also identified on Amberlite columns by Byrd and Kasinsky (1973). Three Hl histones may be recognized in oocytes and postblastula embryos, as well as erythrocytes, although Hla and Hlc. are resolved only on the best gels. At early developmental stages the Hl* spot is always more heavily labeled by [“Hllysine than HUB & c. Carrier histones HL, and HUH & c added to the homogenate show the same recovery, as judged by staining. Erythrocyte histones analyzed alone yielded H~.L, and Hle & c Coomassie blue spots of comparable intensity. In the egg and early cleavage stages, only trace labeling of Hl histones may be seen. This result was also reported by Adamson and Woodland (1974), using 0.4 N HSO, and 5% perchloric acid extraction media. At the blastula stage radioactive Hl spots, although clearly seen, are always rather smeared. The marker Hl histones form dis-

TABLE 1 EFFECT OF EGGS ON RECOVERY OF HI HISTONES FROM LATER-STAGE EMBRYOS” ~Stage Total [“HIcpm in spots of 21) gel HI lysine in~ H2A + H2B jetted Hl H3 H2A + H2B + H:l (cpm). x lo- ’ blastulae blastulae eggs

+ unin-

4.5 4.2

10,549 11,121

14,127 17.110

3,263 7.048

0.61 0,‘s

gastrulae gastrulae eggs

+ unin-

10.9 11.8

55,784 51,530

54,120 41,832

34,052 21,148

0.63 0.8%

” Eight embryos injected with [:‘H]lysine (experiment 1, labeled at stage 8 and frozen at stage 10’~; experiment 2, labeled from stage 9 to stage 11) were homogenized in 40% ethanol, 107 guanidinium chloride and divided into two. Ten unfertilized eggs were added to one half and rehomogenized. The homogenates were processed in the usual way and the histones were fractionated on 2D gels: 6.25 M urea, 0.9 M acetic acid in the first dimension and 18% acrylamide, SDS in the second.

226

DEVELOPMENTAL

BIOLOGY

VOLUME 75,198O

TABLE 2 RECOVERY OF ‘251-H~~~~~~ FROM HOMOGENATES OF OOCYTES, EGGS, AND EMBRYOS” % Recovery Hl Oocyte

Egg Stage 9 blastula Average

15 28 20 23 25 19

Av.

H3

21.5

19 1g

21.5 22 22

38 26 46 33

Av. 19 32 39.5

H2B 4”;

Av. 40.5

H2A ;;

2 ;;

30

41.5 45

4”;

Av.

H4

Av.

Total

Av.

40.5

39 34

36.5

3”;

36

52.5

56 84

70

54 49

51.5

63

54 37

45.5

47

s”i;

47

57

44

n Calf thymus histone (50 pg), containing a trace amount of ““I-calf thymus histone, was homogenized with 10 oocytes, eggs, or embryos in 2 ml of 40% ethanol, 10% guanidinium chloride and processed in the usual way. The histones were fractionated on an 18% acrylamide/SDS gel, and the appropriate stained bands cut out to determine radioactivity.

tinct spots on the same gels. Posttranslational modifications, like phosphorylation, probably produce the smearing. This prevents the resolution of the three Hl species. Recovery of Hl Developmental

Histones Stages

from Different

The low level of Hl synthesis seen in eggs and cleaving embryos could result from preferential loss of this histone during extraction. We have checked that this does not happen by cohomogenizing eggs and radioactively labeled blastulae, and also by adding radioactive histones to homogenates of oocytes, eggs, or blastulae. Table 1 shows the results of adding nonlabeled eggs to blastulae injected with [3H]lysine. Clearly eggs do not cause blastula Hl histones to be lost during extraction and electrophoresis. Table 2 shows the recovery of all histone classes measured by adding 1251-histone to the homogenate. Although the various histone classes apparently varied in the efficiency with which they were extracted, the efficiency was not lower with eggs than with other stages. In this experiment Hl was recovered equally efficiently from all stages, but in a wider range of experiments a little more variation was encountered. H4 was poorly recovered in some experiments with 40% ethanol/lo% guanidium chloride, and Hl showed low recoveries in occasional samples (as can be seen in Fig. 2). In this

experiment iodinated calf thymus histone was used. We have obtained comparable results with lz51-Xenopus histones. Rates of Hl Synthesis opmental Stages

at Different

Devel-

In lysine incorporation experiments the rate of synthesis of a protein may be determined by multiplying the percentage lysine incorporation per hour by a constant, as described in the Methods section. This constant allows for the number of lysine residues per molecule, the molecular weight of the protein, its recovery during extraction, and the size of the lysine pool. The changes in proportion of the injected lysine incorporated per hour into Hl and other histones at egg and later stages are shown in Fig. 2. Variability in the results is seen, and in particular there are occasional samples where the recovery of Hl is low. These ratios are converted into rates of synthesis in Table 3, and data on oocytes are added. It seems that eggs and cleaving embryos make Hl histones at about the same rate as, or only a little faster than, oocytes. Hl histone synthesis increases during the blastula stage, whereas other histones show their most abrupt increase at oocyte maturation, as reported by Adamson and Woodland (1977). Table 4 shows the proportion of total histone synthesis made up by Hl. In the oocyte it is about one-tenth; at maturation it falls and re-

FLYNN

+--cc-

Hl

300

227

Histone HI Synthesis in Carl? Der~clopmenl

ANI) WOODLAND

200

“0 ‘;; I

100

.-‘k

E VI t -6 3 4 .G E VI t

c)-

$

:

0

p

12oc

I-

t3oc I-

4oc

5 1 ‘F

C)-

0 I

I

I

I

I

I

I

2

7

9

10

11

12 5

18

10

20

30

h

Stage

FIG. 2. Hates of histone synthesis from cleavage to the early neurula stage. Incorporation into Hl histone was quantified by cutting spotsout of gels like those shown in Fig. 1, and expressed as explained in the Methods section. The ordinate is proportional to the absolute rate of histone synthesis, a conversion is made for these and other data in Table 3. The horizontal bars represent labeling times and not standard deviations.

mains low during cleavage. Then it rises again to one-tenth-one-fifteenth in the blastula. l~ISCUSSION

The experiments described above confirm the conclusion that Hl synthesis proceeds only at a low rate in unfertilized eggs

and during early cleavage (Adamson and Woodland, 1974). This could have been a recovery artifact, but three lines of evidence argue against this possibility. First, added ‘““I-histone was recovered no more efficiently from eggs than from more advanced embryos. Second, mixing eggs with blastulae did not change the recovery of newll,

228

DEVELOPMENTAL BIOLOGY

synthesized blastula Hl histones, nor did added blastulae increase the recovery of egg Hl histones (unpublished). A third, and even stronger, line of evidence comes from the injection of sea urchin histone mRNA (Woodland and Wilt, 1980a, b). When this mRNA was injected into oocytes or eggs, equally abundant sea urchin Hl synthesis was seen. In the eggs little Xenopus Hl synthesis was detected, in the very same cells that made amounts of sea urchin Hl equivalent to those of the other sea urchin histone classes. If the eggs were fertilized, the Xenopus Hl synthesis rose slowly while that of the sea urchin declined, presumably TABLE

3

RATES OF HISTONE SYNTHESIS AT DIFFERENT DEVELOPMENTAL STAGES” Stage

pg made per hr HI

Large oocyte Expt 1 Expt 2

Expt 3 Mean Unfertilized 2-4 cell + stage 7 morula Stage ‘i-stage 8 blastula Stage &stage 9 blastula Stage g-stage 11 blastula-gastrula Stage ll%-stage 13 gastrula Stage 13/14-stage 18 gastrula-neurula

Total histone 111 187,46, 30

18, 33, 17 20, 19, 6, 7, 12, 9, 10, 2, 7, 7, 5, 6, 7 15, 7, 11, 11, 14, 10 11.5 + 1.5 28,44 46, 25

2105, 1737 2129, 1961

100

1859

160,320

3368, 3207

699, 358

5191,3254

780, 154

3106, 2593

944,269

2394,2442

130

n These rates were calculated from incorporation data as described in the Methods section. The data for stages later than oocytes were the same as those used to prepare Fig. 2. The oocyte data represent measurements using three different females. The mean and the standard error of the mean are shown for the oocytes.

VOLUME X5,1980 TABLE

4

PROPORTION OF HISTONE SYNTHESIS AS HI AT DIFFERENT DEVELOPMENTAL STAGES” Stage Large oocyte Mean Oocyte matured in vitro Unfertilized egg Mean 2/4 cell-stage 7 Mean Stage 7-stage 8 Stage 8-stage 10 Mean Stage g-stage 11 Mean Stage IlM-stage 13 Mean Stage IO-stage 16 Mean Stage 12/13-stage 18 Mean

Ratio of HI to total histone synthesis 0.15,0.11,0.13,0.07,0.05 0.10 +- 0.02 0.03 0.01, 0.03, 0.03, 0.04 0.03 -c 0.006 0.03, 0.02, 0.01 0.02 -+ 0.006 0.05 0.20, 0.44, 0.44, 0.09 0.29 + 0.09 0.13, 0.11 0.12 0.25, 0.06 0.16 0.09, 0.14 0.12 0.39, 0.11 0.25

” These ratios were calculated from the data shown in Table 3 and also from other experiments. The ratios are of masses made, not of lysine incorporation.

because the mRNA was progressively degraded. It seems unlikely that sea urchin and Xenopus Hl histones would display great differences in their recovery or degradability properties, so these observations suggest very strongly that the rate of Xenopus Hl synthesis is low in the egg and rises during late cleavage. In the experiments reported here we have measured recoveries in model experiments and then assumed that these values always pertain. In fact this seems to have been true in a wide range of previous experiments, and examination or scanning of stained marker proteins in the experiments reported here indicated that recoveries did not vary greatly. Two-thirds of the non-H1 histone used up to the blastula stage is made in oogenesis (Woodland and Adamson, 1977). Since we now find that oocytes make Hl in oogenesis, the nuclei assembled during cleavage should not be wholly deficient in Hl his-

FLYNNAND~OODLAND

Histone HI Synthesis in Early Der)elopment

tone. We find somewhat less Hl made in oocytes than might be expected when compared to the other histones. Panyim and Chalkley (1969) found that Hl was 20% of the histone in calf thymus, whereas we find it to be about 10% of the total histone synthesis in oocytes (Table 4). However, our results are insufficiently accurate for this difference to be reliable. Nevertheless, cleavage nuclei are large structures with a diffuse appearance (Graham et al., 1966). This is what would be predicted for Hldeficient nuclei, from the current knowledge of the role of Hl in chromosome structure (see review by Felsenfeld, 1978). Resolution of this point depends on the isolation of Hl histone from the purified chromatin of early embryos. Unfortunately this is an extremely difficult task because nuclei are so few and fragile in early embryos (at the lOO-cell stage there would be no more than 120 pg of Hl histone assembled into chromatin, whereas there are about 25pg of soluble protein and 250 pg of total protein in the embryos; i.e., there would be a 2 X lo:‘- to 2 X lo”-fold excess of non-H1 protein to be removed in the purification). Newly synthesized Hl histone has been isolated from the chromatin of early blastula (stage 7) embryos by Destree (1975). This is technically possible because a much higher proportion of the newly synthesized protein is histone than is true of bulk, stored proteins. However, the amount of Hl histone recovered from the chromatin was small compared to that of the other histones. It is consistent with the amount of Hl histone synthesis that we detected in whole embryos by this stage. Destree et al. (1973) examined bulk chromatin histone a little later, in the stage 9 blastula. In both late blastulae and gastrulae the amount of HI is lower than expected, and it returns to the expected proportion of histones by the neurula stage. A similar rise in Hl has been reported in developing newt embryos (Imoh, 1977, 1978). These results are all consistent with the idea that late blastula

229

nuclei have lower Hl levels than found at later developmental stages. Further work is needed to confirm this postulate. There are three Hl proteins resolved on 2D gels. These proteins are probably different gene products, since similar proteins were seen in newly synthesized molecules programmed in vitro by embryonic mRNA (Ruderman et al., 1979). In the erythrocytes we find approximately equal amounts of HIA and Hle & C, as judged by scanning stained spots. The same result was reported previously for erythrocytes and liver cells by Destree et al. (1973). Using Amberlite chromatography, Byrd and Kasinsky (1973) found three Hl histones in embryos, but these may not be identical to the three species seen by gel electrophoresis. Erythrocytes also contain an H5 histone (Destree et al., 1972; Woodland and Adamson, 1974), which is comparable in amino acid composition to that of birds (Destree, 1975). In early development we find that Hlh is the most abundant newly synthesized species. This correlates with the observation of Destree et al. (1973) that it is also the most abundant species in stained histones from late blastula, gastrula, and neurula chromatin. As far as they go, our results certainly suggest that Xenopus Hl histones do not show a switch in type during development, as is seen in sea urchins (Scale and Aronson, 1973; Ruderman and Gross, 1974; Arceci et al., 1976). We have not found developmental histone changes in Xenopus, even using Triton/urea gels (unpublished results). The same result has also been reported in Triturus embryos (Imoh, 1978). The low rate of Hl histone synthesis in eggs and cleaving embryos is surprising, since the Hl mRNA content, like that of other histones, remains roughly constant from the full-grown oocyte to the blastula stage (Ruderman et al., 1979). The other histones increase their rate of synthesis markedly at maturation of the oocyte, and this must be by posttranscriptional control (Woodland and Adamson, 1977; Ruderman

230

DEVELOPMENTAL BIOLOGY

VOLUME 75,198O

et al., 1979). The constancy of the Hl mRNA content suggests that the later rise in Hl is also mediated by a posttranscriptional control process. Using interspecies, androgenetic, haploid hybrids, Woodland, Flynn, and Wyllie (unpublished) have confirmed that the increase in Hl synthesis is brought about by the mobilization of stored “maternal” mRNA. We conclude that the mechanisms which regulate translation of the maternal store of Hl and non-H1 histone mRNA are partly or wholly distinct for the two classes. We report elsewhere that other stored mRNAs, such as those for the cytoskeletal actins, behave in yet another way (Ballantine et al., 1979). Protein synthesis in early amphibian development is thus controlled by complex posttranscriptional mechanisms. At this time the mass of egg cytoplasm is so large that the few nuclei present cannot exert a quantitatively significant impact on early development through new gene activity.

DESTR~E, 0. H. J., D’ADELHART-TOOROP, H. A., and CHARLES, R. (1972). Analysis of histones from different tissues and embryos of Xenopus laevis (Daudin). I. Technical problems in the purification of undegraded native total histone preparations‘dcta Morphol. Neerl. &and. 10.233-248. DESTRBE, 0. H. J., D’ADELHART-TOOROP, H. A. and CHARLES, R. (1973). Analysis of histones from different tissues and embryos of Xenopus laevis (Daudin). II. Qualitative and quantitative aspects of nuclear histones during early stages of development.

We thank Elizabeth Ballantine and Alison Wyllie for their help. The work was supported by the Medical Research Council.

RUDERMAN, J. V., and GROSS, P. R. (1974). Histones and histone synthesis in sea urchin development.

REFERENCES ADAMSON, E. D., and WOODLAND, H. R. (1974). Histone synthesis in early amphibian development: Histone and DNA synthesis are not co-ordinated. J.

Mol. Biol. 88, 263-285. ADAMSON, E. D., and WOODLAND, H. R. (1977). Changes in the rate of histone synthesis during oocyte maturation and very early development of

Xenopus laevis. Develop. Biol. 57, 136-149. ARCECI, R. J., SENGER, D. R., and GROSS, P. R. (1976). The programmed switch in lysine-rich histone synthesis at gastrulation. Cell 9, 171-178. BALLANTINE, J. E. M., WOODLAND, H. R., and STURGESS, E. A. (1979). Changes in protein synthesis during the development of Xenopus laevis. J. Embryol. Exp. Morph. 51, 137-153. BYRD, E. W., and KASINSKY, H. E. (1973). Histone synthesis during early embryogenesis in Xenopus Zaevis (South African clawed toad). Biochemistry 12, 246-253. DESTR~E, 0. H. J. (1975). “Histones in Development of Xenopus laevis,” Doctoral thesis. University of Amsterdam.

Cell Different. 2, 229-242. FELSENFELD, G. (1978). Chromatin. Nature 271, 115122. GRAHAM, C. F., and MORGAN, R. W. (1966). Changes in the cell cycle during early amphibian development. Develop. Biol. 14.439-460. IMOH, H. (1977). Changes in Hl histone during development of newt embryos. Exp. Cell Res. 108,57-62. IMOH, H. (1978). Re-examination of histone changes during development of newt embryos. Exp. Cell Res. 113, 23-29. NIEUWKOOP, P. D., and FABER, J. (1956). “Normal Table of Xenopus laevis (Daudin).” North-Holland, Amsterdam. PANYIM, S., and CHALKLEY, R. (1969). The heterogeneity of histones. I. A quantitative analysis of calf thymus histones in very long polyacrylamide gels.

Biochemistry 8,3972-3979. Develop. Biol. 36,286-298. RUDERMAN, J. V., WOODLAND, H. R., and STURGESS, E. A. (1979). Histone messenger RNA during the early development of Xenopus laevis. Develop. Biol. 71, 71-82. SEALE, R. L., and ARONSON, A. I. (1973). Chromatinassociated proteins of the developing sea urchin embryo. II. Acid soluble proteins. J. Mol. Biol. 75, 647-658. SHIH, R. J., O’CONNOR, C. M., KEEM, K., and SMITH, L. D. (1978). Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos. Develop. Biol. 66, 172-182. WOODLAND, H. R., and ADAMSON, E. D. (1977). The synthesis and storage of histones during the oogenesis of Xenopus laevis. Develop. Biol. 57, 118-135. WOODLAND, H. R.; and WILT, F. H. (1980a). The functional stability of sea urchin histone mRNA injected into oocytes of Xenopus laevis. Develop. Biol. 75, 199-213. WOODLAND, H. R., and WILT, F. H. (1980b). The stability and translation of sea urchin histone messenger RNA molecules injected into Xenopus laevis eggs and developing embryos. Develop. Biol. 75, 214-221.