Developmental study of the structure of sea urchin embryo and sperm chromatin using micrococcal nuclease

139

Biochimica et Biophysica Acta, 475 (1977) 139--151 © Elsevier/North-Holland Biomedical Press

BBA 98862

DEVELOPMENTAL STUDY OF THE STRUCTURE OF SEA URCHIN EMBRYO AND SPERM CHROMATIN USING MICROCOCCAL NUCLEASE

L. DARWIN KEICHLINE and PAUL M. WASSARMAN * Department of Biological Chemistry and Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Mass. 02115 (U.S.A.) (Received August 24th, 1976)

Summary Sea urchin embryo chromatin is hydrolyzed by micrococcal nuclease into a series of oligomers which are multiples of a monomer (repeating unit) containing 220 + 22 nucleotide pairs of DNA which accumulates during the initial phase of the digestion. Although the size of the chromatin monomers remains the same throughout early development, from the morula through the pluteus stage of embryogenesis, the rate and extent of solubilization of chromatin DNA by micrococcal nuclease .decrease as development proceeds. Sea urchin sperm chromatin is hydrolyzed by micrococcal nuclease into a series of oligomers which are multiples of a monomer containing 260 + 26 nucleotide pairs of DNA which accumulates during the initial phase of the digestion. Analysis of the sizes of oligomers which result from micrococcal nuclease digestion of mouse liver, sea urchin embryo, and sea urchin sperm chromatin in situ, suggests that the oligomers are nearly exact multiples of the respective monomers. These results are discussed in relation to those studies which have shown that the histone complement of the sea urchin embryo and sperm changes during development.

Introduction

Digestion of plant, animal or yeast chromatin with either an endogenous Ca2÷, Mg2÷
140

ture composed of subunits, or "nucleosomes" [3], resembling a string of beads. The protein-to-DNA ratio in isolated nucleosomes is approximately 1.2 : 1 with nearly all of the protein accounted for as the four major types of histones, H2A, H2B, H3 and H4, present in equal amounts [2,3,12]. Reconstitution of bare DNA with only these four histones is sufficient to impart a periodic structure resembling that of native chromatin as evidenced b y X-ray [13] and electron microscopic [3] analyses of the reconstituted material. It is n o w well d o c u m e n t e d that stage-specific changes in histone synthesis take place during early development of the sea urchin; these changes are reflected both qualitatively and quantitatively in the histone complement of developing sea urchin embryos [14--16]. Since histones are responsible for the periodic nature of chromatin, it might be expected that changes in the cellular c o m p l e m e n t of histones during sea urchin embryogenesis would affect chromatin structure. Similarly, the reported presence of one or more unusual histones in sea urchin sperm [17] would be expected to affect the structure of sperm chromatin. In this report we examine the structure of sea urchin e m b r y o and sperm chromatin b y studying the kinetics and products of micrococcal nuclease digestion of chromatin. Materials and Methods

Exogenous nuclease. Micrococcal nuclease (6000 units/mg) was obtained from Worthington Biochemical Corp. and was used without further purification. Preparation o f sea urchin embryo nuclei and chromatin. Gametes of Arbacia punctulata * (Marine Biological Laboratory, Woods Hole, Massachusetts) were collected following coelomic injection with a small volume of 0.5 M KC1. Eggs were washed several times b y settling in filtered artificial sea water (Aquarium Systems, Inc., Eastlake, Ohio) containing antibiotics (penicillin G 100 units/ ml; streptomycin 50 #g/ml) and were finally suspended at a concentration of 4 • 104 per ml and fertilized with freshly diluted semen at 20°C. Embryos were harvested at various stages of development for these experiments only when fertilization rates greater than 95% were obtained. Nuclei were isolated from sea urchin e m b r y o s essentially in the manner described b y Seale and Aronson [18] at 4°C and were checked for cytoplasmic contamination by phase-contrast microscopy. After being washed in 80 mM NaC1, 20 mM EDTA, 10 mM Tris • HC1, pH 7.9, the nuclei were either retained intact for in situ nuclease digestion or were lysed in distilled water using a glass-teflon homogenizer for preparation of chromatin. Chromatin was centrifuged through 1.6 M sucrose, 1 mM EDTA, 10 mM Tris • HC1, pH 7.3, at 1 0 0 0 0 0 X g and the chromatin pellet was dispersed in distilled water at 4 ° C. Preparation o f sperm chromatin. Sperm were prepared for nuclease digestion b y first washing twice in Tris/NaC1/EDTA buffer, pH 7.9. The sperm were then homogenized using a Dounce homogenizer in 1.0 M sucrose, 5 mM EDTA, 80 mM NaC1, 2% Triton X-100, 10 mM Tris • HC1, pH 7.9, and centrifuged at * V i r t u a l l y i d e n t i c a l r e s u l t s w e r e o b t a i n e d using Strongylocentrotus purpuratus (Pacific B i o m a r i n e , Venice, California) e m b r y o s .

141 2000 X g for 30 min. The pellet was re-homogenized in the same buffer. The homogenate was then layered over 2.0 M sucrose, 80 mM NaC1, 5 mM EDTA, 10 mM Tris • HC1, pH 7.5, and centrifuged for 20 min at 1 4 0 0 0 Xg. The pelleted sperm heads were washed three times in Tris/EDTA/NaCI buffer and once in 10 mM MgC12, 1 mM CaC12, 10 mM Tris • HC1, pH 7.4 prior to in situ digestion of chromatin. Alternatively, the sperm heads were lysed in distilled water resulting in a highly viscous chromatin gel. Preparation of mouse liver nuclei. Nuclei were isolated from livers of adult Swiss mice (CD-1, Charles River Laboratories, Wilmington, Massachusetts) using a modification of the procedure described by Hewish and Burgoyne [1]. All preparative steps were carried o u t at 4 ° C. Fresh tissue was homogenized in a buffer containing 2.2 M sucrose, 25 mM KC1, 2 mM MgC12, and 10 mM Tris • HC1, pH 7.2, using a Polytron PT-10 homogenizer. Nuclei were pelleted by centrifugation at 3 7 0 0 0 X g for 1 h. The pelleted nuclei were washed once in the homogenization buffer brought to 0.5 M sucrose and 0.1% Triton X-100, and were then washed several times with 80 mM NaC1 and 20 mM EDTA adjusted to pH 7.2, using gentle homogenization in a glass-teflon homogenizer to resuspend the nuclear pellet. Digestion of chromatin. Chromatin was digested in situ b y incubating isolated nuclei at 37°C in a buffer containing 1 mM CaC12, 10 mM MgC12, and 10 mM Tris • HCI, pH 7.4 in the presence of micrococcal nuclease. Isolated chromatin was digested under the same conditions. Endogenous nuclease activity was assessed b y incubation of nuclei or isolated chromatin under identical conditions, b u t in the absence of micrococcal nuclease. Digestions were terminated for electrophoretic analysis of DNA fragments by the addition of nine-volumes of a solution containing 5 mM EDTA, 1.1% sodium dodecyl sulfate, and 15 mM Tris • HCI, pH 9.0, followed by gentle mixing. The solution was brought to 1 M in NaC1 and extracted with phenol. The aqueous phase was dialyzed overnight against distilled water and then lyophilized. DNA was dissolved in a small volume of water and its concentration was determined either b y the diphenylamine procedure of Burton [19] or by absorbance at 260 nm. For analysis of DNA solubilization, digestions were terminated at appropriate times by the addition of cold 80 mM NaC1, 20 mM EDTA, pH 7.3, followed immediately b y chilling the tubes at 4 ° C. The digests were then centrifuged at 13 000 × g for 10 min and the supernatants were carefully drawn off and analyzed either spectrophotometrically at 260 nm or by liquid scintillation counting. In some cases, the percentage of chromatin rendered acid-soluble b y nuclease digestion was determined by precipitation of DNA with 0.17 M HClO4, centrifugation, and measurement of absorbance of the supernatant at 260 nm. Electrophoretic analysis of DNA fragments. DNA fragments isolated from digested chromatin were analyzed by electrophoresis on 3.0% polyacrylamide slab gels containing 0.5% agarose. The gels were made up in 0.1 M Tris/borate buffer, pH 8.3, containing 0.25 mM EDTA. Samples containing 5--15/~1 were applied to the gels. Gels were stained with 0.001% ethidium bromide for approximately 2 h to identify DNA bands and were photographed under ultraviolet light using a Kodak 23A filter. Gels were calibrated with DNA fragments of defined length prepared from an endo R • Hind III digest of SV40 DNA [20]. Photographic negatives of stained gels were scanned using an Ortec microdensitometer model 4300.

142

Results

Solubilization o f sea urchin embryo chromatin DNA by micrococcal nuclease The rate and extent of digestion of sea urchin embryo chromatin by micrococcal nuclease are correlated with the stage of embryonic development at which the chromatin is isolated. Fig. 1 shows relative rates and extents of solubilization of chromatin DNA isolated at several stages of sea urchin embryogenesis: morula, hatching blastula, mesenchyme blastula, gastrula, and pluteus. In each case, 250 #g/ml of chromatin DNA, labelled with [3H]thymidine, was incubated at 37°C in the presence of 12.5 units/ml of micrococcal nuclease for up to 3 h. Chromatin isolated from the two earliest developmental stages, morula and hatching blastula, is digested very rapidly by micrococcal nuclease, with 50% of the chromptin DNA solubilized in approximately 10 min and more than 70% solubilized in 2 h. By comparison, the rates and extents of digestion of sea urchin chromatin decrease progressively as development proceeds through mesenchyme blastula, gastrula, and pluteus (Table I). For example, even after 2 h of incubation in the presence of micrococcal nuclease, pluteus chromatin DNA, like mouse liver chromatin DNA [8], has not exceeded 50% solubility.

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143 TABLE I RATE AND EXTENT COCCAL NUCLEASE

OF SOLUBILIZATION

OF SEA URCHIN

CHROMATIN

DNA BY MICRO-

Source of chromatin (embryonic Stage)

Time to achieve 50% solubilization tion of DNA (rain) *

Rate of s o l u b i l l z a t i o n relative t o m o u s e liver chromatin * *

Extent of solubUization o f D N A (%) *

Morula Hatching blastula Mesenehyme blastula Gastrula Pluteus

10 9 35 53 60

6.0 6.7 1.7 1.1 1.0

70--80 70--80 55--65 45--55 40---50

* D a t a o b t a i n e d l ~ o m e x p e r i m e n t s p e r f o r m e d in a m a n n e r i d e n t i c a l t o t h o s e s h o w n in Fig. 1. ** T i m e ( r a i n ) n e c e s s a r y t o a c h i e v e 5 0 % s o l u b i l i z a t l o n o f m o u s e Hver e l ~ o m a t i n D N A i n t h e p r e s e n c e o f m i e r o c o c c a l n u c l e a s e as c o m p a r e d t o sea u r c h i n e m b r y o e h r o m a t i n D N A u n d e r c o m p a r a b l e c o n ditions.

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DIGESTION TIME (rain) Fig. 2. Production o f soluble nuelaofldas b y endogenous endonuelaase associated with e ~ o m a U n isolated f r o m sea urchin embryos at several stages o f development. Embryos were labeled wit~ [ 3 H ] t h y m l c l i n e as in Fig. 1. C h r o m a t i n w a s i s o l a t e d iLrom s e a u r c h i n e m b r y o s a t m o r u l a (__-), h a t c h i n g b l a s t u l a (¢ .~), m e s e n c h y m e b l a s t u l a ( ~ A ) , g a s t r u l a (4 ~), a n d p l u t e u s (o o), and incubated u p t o 1 8 0 mill w i t h o u t a d d e d n u e l e a s e a t 3 7 ° C . D i g e s t i o n s w e r e t e r m i n a t e d , t h e s a m p l e s w e r e c e n t r l l u g e d 0 a n d t h e s u p e r n a t a n t w a s a s s a y e d as d e s c r i b e d i n M a t e r i a l s a n d M e t h o d s .

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0 Fig. 3. E l e c t r o p h o r e t i c analysis of D N A p r e p a r e d f r o m m o u s e liver c h r o m a t i n . D N A was p r e p a r e d f r o m n u c l e i i n c u b a t e d for 15 rain in t h e p r e s e n c e of 4 5 u n i t s ] m l of m i c r o c o c c a l n u c l e a s e a n d 5 ttg was subj e c t e d t o e l e c t r o p h o r e s i s o n a 3,0% p o l y a c r y l a m i d e gel. T h e e x p e r i m e n t a l c o n d i t i o n s a n d e l e c t r o p h o r e t i c p r o c e d u r e are d e s c r i b e d in Materials a n d M e t h o d s . A p h o t o g r a p h i c n e g a t i v e of t h e e t h i d i u m b r o m i d e s t a i n e d gel was s c a n n e d using an O r t e c m i c r o d e n s i t o m e t e r . M i n d i c a t e s m a i n - b a n d D N A a n d n u m b e r s [ 1 ] t h r o u g h [6] i n d i c a t e m o n o m e r t h r o u g h h e x a m e r , r e s p e c t i v e l y .

cally into a series of oligomers representing multiples of the smallest DNA fragment, containing approximately 200 nucleotide pairs, which accumulates during the course of the digestion. Such an electrophoretic pattern, together with a densitometer tracing, is shown in Fig. 3 for mouse liver chromatin which has undergone moderate digestion with micrococcal nuclease in situ (within nuclei). In addition to a main-band [M] of high molecular weight DNA, a series of .DNA fragments are readily distinguishable which increase in size from the m o n o m e r [1], containing approximately 200 nucleotide pairs, to the hexamer [6], containing approximately 1200 nucleotide pairs. Electrophoresis has been used to compare the sizes and amounts of DNA fragments produced during micrococcal nuclease digestion of chromatin in situ or isolated from the sea urchin at several stages of embryonic development. In Fig. 4 the electrophoretic patterns of DNA fragments, together with densitometer tracings, are shown for sea urchin chromatin from embryos at hatching blastula and pluteus incubated in situ with 2.5 units/ml of micrococcal nuclease for 2 min and 5 min at 37°C. Whereas the undigested chromatin preparations contain primarily high molecular weight main-band DNA [M] (Fig. 4a), the digested chromatin samples from both hatching blastula and pluteus stage embryos show a series of oligomers which represent multiples of the smallest DNA fragment containing 220 -+ 22 nucleotide pairs (Fig. 4c). After 2 min of incubation in the presence of micrococcal nuclease, the main-band of hatching blastula chromatin DNA displays appreciable broadening toward lower molecular weights, and in addition, discrete bands of DNA, as small in size as 220 +

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Fig. 4. E l e c t r o p h o r e t i c a n a l y s i s o f D N A p r e p a r e d f r o m sea u r c h i n e m b r y o s a t h a t c h i n g b l a s t u l a o r p l u t e u s stage. DNA was prepared from hatching blastula ( ) or pluteus (...... ) n u c l e i i n c u b a t e d for (a) z e r o , (b) 2 m i n , o r (c) 5 r a i n in t h e p r e s e n c e o f 2 . 5 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e . E a c h s a m p l e a p p l i e d t o t h e 3 . 0 % p o l y a c r y l a m i d e gel c o n t a i n e d 5 Dg o f D N A . T h e e x p e r i m e n t a l c o n d i t i o n s a n d e l e c t r o p h o r e t i c p r o c e d u r e a r e d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e m e t h o d o f a n a l y s i s is d e s c r i b e d i n Fig. 3.

22 nucleotide pairs, are distinguishable. On the other hand, under the same conditions, chromatin from pluteus stage embryos undergoes significantly less digestion in situ, with most of the DNA remaining as a discrete band of high molecular weight material (Fig. 4b). Incubation of hatching blastula chromatin in the presence of micrococcal nuclease for 5 min results in the conversion of nearly all main-band DNA into smaller oligomers, with nearly one-third of the DNA migrating as the monomer containing 220 + 22 nucleotide pairs; less than 10% of the chromatin DNA has been solubilized during this incubation period. In the same period of time, pluteus chromatin has also undergone a significant amount of digestion into smaller discrete fragments; however, as shown in Fig. 4c, the extent of digestion for pluteus chromatin is considerably less than that for hatching blastula chromatin under the same conditions. It should be noted that the differences observed between the digestibility of hatching blastula and pluteus chromatin in situ, are also observed using isolated chromatin.

Analysis o f DNA fragments after micrococcal nuclease digestion o f sea urchin sperm chromatin Electrophoresis has also been used to examine the sizes of DNA fragments produced during the incubation of isolated sea urchin sperm chromatin or sperm chromatin in situ with micrococcal nuclease. Digestion of sperm chromatin in situ at 37°C with 25 units/ml of micrococcal nuclease for 2 min results in the conversion of a large amount of main-band DNA into a series of oligomers representing multiples of the smallest DNA fragment containing 260 + 26 nucleotide pairs; similar results were obtained using isolated sperm chromatin. A comparison of the electrophoretic mobility of sperm DNA fragments with mouse liver DNA fragments is presented in Fig. 5. The 260 + 26 nucleotide pair fragment of sperm chromatin accumulates during the initial phase of the digestion and is degraded to smaller pieces only after prolonged incubation under these conditions or upon exposure to much higher concentrations of micrococcal nuclease.

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0 F i g . 5. E l e c t r o p h o r e t i c a n a l y s i s o f D N A p r e p a r e d f r o m m o u s e liver n u c l e i o r s e a u r c h i n s p e r m . T o p : D N A w a s p r e p a r e d f r o m liver n u c l e i i n c u b a t e d f o r 1 5 m i n in t h e p r e s e n c e o f 4 5 u n i t s / m l o f m i c r o c o c c a l n u c l e ase a n d 5 /~g w a s s u b j e c t e d t o e l e c t r o p h o r e s i s o n a 3 . 0 % p o l y a c r y l a m i d e gel. B o t t o m : D N A w a s p r e p a r e d f r o m sea u r c h i n s p e r m i n c u b a t e d f o r z e r o ( c o n t r o l ) o r 2 r a i n in t h e p r e s e n c e of 2 5 u n i t s / m l o f m i c r o c o c c a l n u c l e a s e . T h e e x p e r i m e n t a l c o n d i t i o n s a n d e l e c t r o p h o r e t i c p r o c e d u r e a r e d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e m e t h o d o f a n a l y s i s is d e s c r i b e d in F i g . 3. L 1 - - L 5 r e f e r t o t h e p o s i t i o n s o f t h e liver m o n o m e r through pentamcr, respectively.

Analysis o f the relationship between chromatin monomers and oligomers •In order to gain additional insight into the structure of sea urchin e m b r y o and sperm chromatin, the data obtained in this study have been analyzed to determine whether the DNA fragments generated by micrococcal nuclease are exact multiples of 220 + 22 and 260 + 26 nucleotide pairs, respectively. This analysis is shown in Fig. 6 and the results are summarized in Table II. The actual mean sizes (from electrophoretic mobility) of the chromatin oligomers

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Fig. 6. Analysis of DNA o]igomer size in mouse liver, sea urchin sperm, and sea urchin hatching blastula e h r o m a t i n . T h e a c t u a l sizes of t h e D N A o l i g o m e r s , e s t i m a t e d f r o m t h e i r e l e c t r o p h o r e t i c m o b i l i t y , are p l o t t e d (o) t o g e t h e r w i t h a n i n d i c a t i o n of t h e e r r o r i n v o l v e d in t h e m e a s u r e m e n t ( ± 1 0 % ) . T h e sizes of t h e s e o l i g o m e r s are c o m p a r e d (a) w i t h t h e p r e d i c t e d sizes o f t h e o l i g o m e r s , a s s u m i n g t h e o l i g o m e r s are e x a c t m u l t i p l e s of t h e r e s p e c t i v e m o n o m e r s ( . . . . . . ) a n d (b) w i t h t h e p r e d i c t e d sizes of the o l i g o m e r s , a s s u m i n g t h e r e is a n " i n t e r - m o n o m e r " r e g i o n of D N A w h i c h c o n t a i n s 20% o f t h e n u m b e r o f n u c l e o t f d e pairs w h i c h m a k e u p t h e m o n o m e r ( . . . . . ).

T A B L E II S U B U N I T SIZES OF C H R O M A T I N FROM V A R I O U S SOURCES Source of c h r o m a t i n

S u b u n i t size ( n u c l e o t i d e pairs o f D N A , -+10%)

Liver (mouse) S p e r m (sea u r c h i n ) H a t c h i n g b l a s t u l a (sea u r c h i n ) P l u t e u s (sea u r c h i n )

2 0 0 * ( 2 1 3 ) ** 260 (258) 220 (233) 220 (228)

* V a l u e d e t e r m i n e d d i r e c t l y f r o m e l e c t r o p h o r e t i c m o b i l i t y of m o n o m e r . ** V a l u e d e t e r m i n e d b y s u b t r a c t i n g t h e l e n g t h s o f n e i g h b o r i n g o l i g o m e r s ( d e t e r m i n e d d i r e c t l y f r o m electrophoretic mobility) f r o m one another and calculating the m e a n difference.

148 isolated from mouse liver, sea urchin sperm, and sea urchin hatching blastula nuclei, following moderate digestion in situ with micrococcal nuclease, have been compared (a) with the predicted sizes of the oligomers based upon exact multiples of the m o n o m e r and (b) with the predicted sizes of the oligomers based upon the possibility of the presence of an "inter-monomer" region (representing 20% of m o n o m e r DNA) [4,8,39,40] (Fig. 6). The results of this type of analysis strongly suggest that the oligomers are, indeed, nearly exact multiples of the respective monomers. Discussion

The interphase chromosome of plants and animals exists as a complex containing equal weights of DNA and histones, together with variable amounts o f non-histone proteins ("chromatin") [22]. Evidence obtained from X-ray, electron microscopic, optical, and h y d r o d y n a m i c measurements suggests that the DNA of eukaryotic chromosomes is highly condensed, largely as a consequence of its interaction with histones, and that chromatin fibers possess a periodic structure [23,24]. Using DNA-specific endonucleases it has been shown that chromatin from sources as diverse as viruses and animals exists as a repeating structure composed of subunits, each of which contains approximately 200 nucleotide pairs of DNA, and which apparently gives rise to the periodic structure of chromatin (refs. 1--8, 25 and Shelton, E., Wassarman, P.M. and De Pamphilis, M.L., unpublished). Recently it has been possible to reconstitute bare DNA with four types of histones, the two lysine-rich histones (H2A, H2B) and the t w o arginine-rich histories (H3, H4), and to obtain material which is hydrolyzed b y micrococcal nuclease into a series of discrete oligomers [26, 41], which exhibits spherical particles [3], and which generates an X-ray diffraction pattern that is virtually identical to that obtained with native chromatin [13]. While the actual composition and arrangement of histones within the chromatin subunit (e.g. refs. 27, 28) is subject to speculation, there seems little d o u b t that it is the histones which impart a periodicity to chromatin. Since it is clear that histones play a fundamental structural role in chromatin, we have studied the structure of chromatin in the sea urchin during early embryogenesis, a period when the cellular histone complement is changing both qualitatively and quantitatively [14--16], as well as the structure of chromatin in sea urchin sperm which are reported to contain a unique histone(s) [17]. We have found that sea urchin chromatin exhibits marked changes in its susceptibility to micrococcal nuclease during early embryogenesis, while maintaining a 220 -+ 22 nucleotide pair subunit at all stages of early development examined. A major change in chromatin structure apparently takes place subsequent to the hatching blastula stage when both a decreased rate and extent of solubilization of chromatin DNA by micrococcal nuclease is manifested. By the time the pluteus stage of embryogenesis is reached, sea urchin chromatin is hydrolyzed by micrococcal nuclease at virtually the same rate and to the same extent as adult mouse liver chromatin. Sea urchin sperm chromatin is hydrolyzed into a series of oligomers which are multiples of a 260 -+ 26 nucleotide pair subunit, however, the kinetics of digestion of sperm chromatin are similar to those obtained with liver chromatin.

149 Investigators in several laboratories have studied histone synthesis during embryogenesis in the sea urchin [14--16]. Despite some earlier reports to the contrary, recent studies have shown that histone synthesis in the sea urchin is initiated as early as the first cleavage division and that newly synthesized histones continually become associated with the embryo's chromatin. On the other hand, there is not complete agreement as to the nature of the histones synthesized or as to their relative rates of synthesis at various stages of embryogenesis in the sea urchin. It would appear that the histones synthesized during cleavage in the sea urchin associate rapidly and stably with chromatin. Cohen et al. [16] found that all histone species remain associated with chromatin even after certain of them cease to be synthesized, and they concluded that such a situation should give rise to multiple forms of nucleosomes, with their exact histone composition dependent upon the particular stage of sea urchin development. Our results indicate that, even if the histone complement of the nucleosomes becomes heterogenous during embryogenesis, the approximate size of the nucleosomes remains constant at 220 + 22 nucleotide pairs of DNA. On the other hand, our observations concerning the changes in susceptibility of sea urchin chromatin to micrococcal nuclease as a function of the stage of embryogenesis could be related, at least in part, to changes in the histone complement during development. For example, nucleosomes composed of those histones characteristic of early cleavage may be more readily hydrolyzed by micrococcal nuclease than nucleosomes formed at gastrula and pluteus. It is of interest that the decrease in the rate and extent of chromatin DNA solubilization by micrococcal nuclease during embryogenesis accompanies the decrease in the incidence of DNA replication and cell division. Several investigators have noted differences in the behaviour of chromatin, presumably due to changes in chromatin structure, during the cell cycle and during embryogenesis, as well. The susceptibility of chromatin to DNAase I digestion [29], the extent of binding of intercalating dyes to chromatin [29,30], the circular dichroism spectrum and thermal stability of chromatin [30], and the degree of interaction of heparin with chromatin [31] are all dependent upon the specific stages of the cell cycle at which the chromatin is examined. All of these criteria suggest that there are significant changes in the organization of chromatin during the cell cycle and that, in particular, chromatin DNA is more accessible during early to mid-S phase. The latter is supported by the finding that chromatin from rapidly growing cells is significantly more susceptible to DNAase I digestion than chromatin from confluent non-growing cells [29]. In this connection, Seale [32] found that newly synthesized DNA became progressively less susceptible to nuclease {DNAase I and micrococcal nuclease) digestion with time. These and other observation~ [33] suggest that newly replicated chromatin DNA may have a reduced protein content in comparison with other regions of chromatin. Accordingly, the stage-specific differences in chromatin DNA susceptibility to micrococcal nuclease which we report here could be related to changes in the cell cycle during embryogenesis [34]. Micrococcal nuclease has been used previously to study the structure of sperm chromatin in two different types of biological systems: trout testis [35], in which there is a switch from nucleohistone to nucleoprotamine during devel-

150

o p m e n t of the sperm, and echinoderm testis [21], in which nucleohistones are maintained in the mature sperm. It would appear that the transition from histone to protamine greatly affects the structure of the testis chromatin as reflected in its lack of susceptibility to hydrolysis by micrococcal nuclease [35]. On the other hand, in sperm which do not undergo a histone-to-protamine transition during development, the tight packing of the chromatin in the mature sperm does n o t restrict its accessibility to attack by micrococcal nuclease [21]. Our results suggest that values for the rate and extent of hydrolysis of sea urchin sperm chromatin, as evidenced by the percentage of DNA solubilization, are n o t unlike those for mouse liver chromatin. We have found that sea urchin sperm chromatin is hydrolyzed by micrococcal nuclease into a series of oligomers which are multiples of a m o n o m e r containing, not 200 nucleotide pairs of DNA, b u t rather 260 + 26. Like the repeating unit of chromatin from other eukaryotic organisms, the 260 nucleotide pair m o n o m e r of sperm chromatin is susceptible to further digestion by micrococcal nuclease and is hydrolyzed into DNA fragments containing from approximately 50 to 170 nucleotide pairs. Perhaps the difference in m o n o m e r size (260 versus 160 nucleotide pairs) reported here, as compared to the report of others [21], can be attributed entirely to the different echinoderm studied or to differences in experimental conditions. Analysis of the sizes of oligomers which result from micrococcal nuclease digestion of mouse liver, sea urchin e m b r y o , and sea urchin sperm chromatin in situ suggests that the oligomers are nearly exact multiples of the respective monomers (Fig. 6). Estimates of the m o n o m e r size were made n o t only on the basis of the electrophoretic migration of the m o n o m e r itself, b u t also by subtracting the lengths of neighboring oligomers from one another [37,38] which virtually eliminates the effect of degradation of the DNA from the ends of the oligomers. It should be noted that these analyses were carried o u t on micrococcal nuclease digests of chromatin which had undergone only moderate hydrolysis; a substantial proportion of the chromatin DNA remained as high molecular weight material and less than 10% of the DNA had been solubilized. These results suggest that all three types of chromatin are organized into regions containing nucleosomes which are arranged contiguously, while other regions, perhaps, do n o t contain nucleosomes. This is not meant to imply that the " n o n n u c l e o s o m e " regions of chromatin are devoid of protein, b u t only that they are much more susceptible to hydrolysis by micrococcal nuclease than regions made up of " t r u e " nucleosomes.

Acknowledgements We are very grateful to Ms. Kim Goh for her contributions to this research and to Dr. Mel De Pamphilis for many enlightening discussions. This research was supported b y grants awarded to P.M.W. b y the National Cancer Institute, DHEW, and b y The National Science Foundation.

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