Assembly of newly replicated chromatin

Cell, Vol. 15. 969-977,

Assembly

November

1976,

Copyright

0 1976 by MIT

of Newly Replicated

A. Worcel, S. Han and M. L. Wong Department of Biochemical Sciences Princeton University Princeton, New Jersey 08540

Summary Mild staphylococcal nuclease digestions under isotonic conditions release fragments of a 200 A diameter fiber from nuclei of Drosophila melanogaster tissue culture cells. These soluble fragments have high sedimentation coefficients (30100s) and show tightly packed nucleosomes in the electron microscope. Under the same conditions, newly replicated chromatin is released as more slowly sedimenting fragments (14s). Within 20 mln after DNA replication, the nascent chromath! gradually matures into compact supranucleosomal structures which are indistinguishable from bulk chromatln on the isoklnetic sucrose gradients. We have used this fractionation technique to examine the question of the fate and assembly of the new histones. After short pulses with either 9-methionine or 3H-lysine, the radioactive histones do not co-sediment with the bulk chromatin but appear instead In the fractions where the newly replicated DNA is found. Furthermore, the various nascent histones appear in different fractions on the gradient: histones H3 and H4 in lo15s structures, histones H2A and H2B in 15-50s structures and histone Hl in 30-100s structures. These results, together with the analysis of pulse and pulse-chase experiments of both nascent DNA and histones, strongly suggest that histones H3 and H4 are deposited first on the nascent DNA (during or slightly after the DNA is replicated), histones H2A and H2B are deposited next (2-10 min later) and histone Hl is deposited last (lo-20 min after DNA replication). A high turnover 20,000 dalton protein is also associated with the newly replicated chromatin. Introduction Although the nucleosomal structure of chromatin is well established (for reviews see Kornberg, 1977; Felsenfeld, 1978), the manner of assembly of the newly replicated chromatin is still poorly understood and controversial. The results of Weintraub (1973) Tsanev and Russev (1974) and Freedlander, Taichman and Smithies (1977) suggest that the newly synthesized histones associate with the newly made DNA. On the other hand, the results of Jackson, Granner and Chalkley (1975, 1976), Seale (1976) and Hancock (1978) seem to indicate that

Chromatin

the new histones distribute themselves randomly among the new and old DNA. In these later studies, the nascent chromatin was fractionated by density labeling the newly made DNA followed by formaldehyde cross-linking and subsequent cesium chloride-banding of the cross-linked nucleoprotein. Since such drastic treatments could cause nucleosome rearrangements, we decided to analyze the assembly of the nascent cellular chromatin using a more physiological chromatin fractionation. We have succeeded in isolating soluble supranucleosomal structures, sedimenting at 50s and above under isotonic conditions, from bulk chromatin of exponentially growing tissue culture cells. We have also found that the newly replicated chromatin has a different conformation and sediments under the same conditions at 14s. Such a fractionation allowed us to examine directly the question of the assembly of the newly made histones into chromatin. Results Isolation of Soluble, Compact Supranucleosomal Structures We have developed a fast procedure for the purification of compacted supranucleosomal structures from nuclei of Drosophila melanogaster tissue culture cells. Our method is a modification of the procedure of Renz, Nehls and Hozier (1977) and involves mild digestions with staphylococcal nuclease under isotonic conditions (see Experimental Proced ures). The solubilized chromatin fragments account for lo-50% of the total chromatin (depending upon the extent of digestion) and sediment, after a mild nuclease digestion, as a broad peak with a maximum at around 50s (see below, Figures 2a and 3a3~). At the concentrations of nuclei used (
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cleosomes (c); and BOS, over 30 nucleosomes (d). The average number of nucleosomes per supranucleosomal cluster can easily be determined by counting the number of nucleosomes after partial unraveling of the unfixed structures (right halfpanels in Figure 1); this number agrees with both the position of the particles in the isokinetic sucrose gradients which resolve the individual oligonucleosome peaks (see below, Figures 2b and 2c) and the expected size of their DNA (as determined by electrophoresis of the phenol-extracted DNA on agarose gels; data not shown). The compact supranucleosomal structures containing 6-14 nucleosomes (Figures la and 1 b, left half-panels) appear as irregularly shaped, globular particles (“superbeads”; Hozier, Renz and Nehls, 1977) of about 200-250 A in diameter. The larger supranucleosomal structures, however, are not globular; they appear to be fragments of a 200 A diameter fiber (Figures lc and Id, left half-panels). The long fragments are gently or sharply bent and sometimes have a knobby appearance suggesting that they may be made up of tightly packed superbeads, although we could detect no regular, uniformly sized superbead units under our experimental conditions. A detailed electron micrograph analysis of the various supranucleosome structures will be published elsewhere (M. L. Wong and A. Worcel, manuscript in preparation). For the purposes of the present work, it suffices to state that the electron microscope studies clearly support the notion that the high sedimentation coefficients of the solubilized chromatin must be due to the compacted nature of the nucleosomal clusters and not to an artifactual aggregation of the chromatin fragments.

Structure of the Nascent Chromatin Nascent chromatin labeled by a short pulse with 3H-thymidine sediments more slowly than bulk chromatin under our experimental conditions. Figure 2 shows the sedimentation profiles of nascent chromatin (labeled with a 20 set pulse with 3Hthymidine) and bulk chromatin (labeled with 14Cthymidine for 24 hr) after various extents of digestion with staphylococcal nuclease. After mild digestions, the nascent chromatin fragments sediment at about 14S, while the bulk chromatin fragments sediment with coefficients of 50s and above (Figure 2a). After more extensive digestions, the sedimentation rate of the bulk chromatin fragments gradually decreases, with the broad 50s peak breaking down first into the familiar oligonucleosome peaks (Finch, Noll and Kornberg. 1975; Nell and Kornberg, 1977) and finally into 11s monosomes. Throughout these marked changes in the sedimentation rate of the solubilized bulk chromatin, the sedimentation rate of the nascent chroma-

tin changes only slightly, with the end result that after extensive digestion, a 14s shoulder is still present on the nascent chromatin profile (Figure 2d). These results indicate that nascent chromatin must have a structure different from the bulk chromatin structure. This peculiar conformation of the nascent chromatin is a transient state. Figure 3 shows the sedimentation profiles of nascent chromatin fragments released after a mild nuclease digestion as a function of time after DNA replication. The sedimentation profile of the bulk chromatin shows the usual broad peak with a maximum at about 50s in the three panels, while the nascent chromatin gradually “matures” into more quickly sedimenting structures; Figures 3b and 3c show the profiles of a 2 min and a 10 min chase, respectively, of the 20 set pulse with 3H-thymidine shown in Figure 3a. By 20 min after DNA replication, the sedimentation profile of the nascent chromatin fragments is indistinguishable from that of bulk chromatin fragments (results not shown). Somewhat similar enrichments for slowly sedimenting nascent chromatin subunits have been reported by Hildebrand and Walters (1976), although the lower sedimentation velocity of their bulk chromatin oligonucleosomes resulted in a less marked fractionation than that reported here. The 14s fractions are enriched in nascent chromatin more than 80 fold vis-a-vis the 50s fractions, as can be ascertained by comparing the 3H/14C ratios on the 14s and 50s peaks in Figures 2a and 3a.

Distribution of Nascent Histones on the Fractionated Chromatin We have examined the assembly of the newly replicated chromatin by pulse-labeling cells with either 35S-methionine or 3H-lysine followed by chromatin fractionation as before and analysis of the proteins in the fractionated sucrose gradients by SDS gel electrophoresis and fluorography. Figure 4 shows a stained gel (a) and the fluorograph of the same gel (b) from an experiment in which the exponentially growing cells were pulse-labeled for 2 min with 35S-methionine immediately before chromatin fractionation as described above. The distribution of the total nonradioactive histones across the gradient closely parallels the DNA profile (the total DNA profile for this experiment, not shown, was very similar to the bulk DNA profile shown in Figure 3), with the concentration of the five histones showing a maximum at around 5OS, as expected. The distribution of the nascent histones, on the other hand, is markedly different. Most of the % radioactivity is found in the more slowly sedimenting fractions. Furthermore, the labeled histones do not all appear in the same fractions. Histones H3 and H4 appear to be depos-

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Figure

of Newly

1. Electron

Replicated

Micrographs

Chromatin

of Isolated

Supranucleosomal

Structures

Aliquots from selected fractions of the isokinetic sucrose gradients were processed for electron microscopy as described in Experimental Procedures, with (left half-panels) or without (right half-panels) previous fixation for 2 min on ice in 1% glutaraldehyde. (a) 30s fraction; (b) 50s fraction; (c) 70s fraction; (d) 90s fraction.

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2. Sucrose

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and Sulk Chromatin

as a Function

of Nuclease

Concentration

Cells were labeled with Y-thymidine for 24 hr and W-thymidine for 20 sec. 2 x 10’ nuclei were incubated for 15 min on ice with (a) 60, (b) 120. (c) 240 and (d) 960 U of staphylococcal nuclease, and sedimented through isokinetic sucrose gradients as described in Experimental Procedures. Sedimentation is from left to right. The numbers within each panel indicate the positions of the resolved bulk oligonucleosome peaks (monomer to decamer).

ited on 10-15s fractions, histones H2A and H2B on 20-50s fractions and histone Hl on 505 fractions. This unusual result is not due to the effects of differently sized histone pools. For instance, a large intracellular pool of cold histones H2A and H2B could have diluted the specific activity of the radioactive H2A and H2B in the 14s fractions (although such pools could still not explain the appearance of labeled H2A and H2B in the 20-50s fractions). Exponentially growing tissue culture cells do not contain appreciable histone pools (Weintraub, 1973). To rule out histone pool size effects, however, we performed experiments using both longer labeling times (to deplete the hypothetical histone pools) and cold chases of the radioactive precursor. Longer labeling times with 35S-methionine do not change the pattern of histone deposition. Figure 5b shows the fluorograph of a

lo-min 35S-methionine pulse fractionated as before (the extent of nuclease digestion and the total DNA and histone profiles across the gradient in this experiment were identical to the ones shown in Figures 3 and 4). The main difference between this experiment (Figure 5b) and the one shown in Figure 4b is that the nascent H3 and H4 histones are not only present in the 10-15s fractions, but can also be found down the gradient all the way into 50s fractions. This result is readily explained by the kinetics of maturation of the nascent chromatin (see Figure 3 above). Despite this longer pulse, the nascent histones H2A and H2B are still found only on 20-60s structures and histone Hl is found mainly on 50s structures. Identical results were obtained using 3H-lysine instead of %-methionine as the pulse label (all five Drosophila histones contain at least one methi-

Assembly 973

of Newly

Replicated

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SEDIMENTATION

COEFICIENT

FRACTION Figure 3. Sucrose matin as a Function

Gradient of Time

(S)

NUMBER

Profiles of Nascent after DNA Replication

and

Bulk

Chro-

2 x 10’ nuclei were incubated with 60 U of staphylococcal nuclease for 15 min on ice. (a) Cells were labeled and processed as described in the legend to Figure 2; (b) 2 min chase of the SHthymidine pulse; (c) 10 min chase of the JH-thymidine pulse. Sedimentation coefficients (S) across the gradients were estimated from both the position of 40s and 60s ribosomal subunits and the position of the Hl-containing oligonucleosomes of known S values (Noll and Kornberg, 1977).

onine residue; Alfageme et al., 1974). Chases of the pulse label (40 min chases in the presence of excess nonradioactive methionine or lysine) or longer labeling times (over 3 hr of continuous labeling with either 35S-methionine or 3H-lysine) gave a distribution of labeled protein across the gradient which was identical to the total protein distribution seen by Coomassie blue staining of the gels. Further proof for the absence of histones H2A and H2B in newly replicated chromatin comes from the protein analysis of chromatin fragments released under extremely mild digestions. Practically no bulk chromatin is solubilized at very low nuclease concentrations (~20 U per 1OB nuclei under our experimental conditions; see Experimental Procedures), although a small but significant fraction (-10%) of the nascent chromatin is solubilized under these conditions. Figure 5a shows the results of such a very mild digestion (after a 10 min pulse with 35S-methionine). The nascent chromatin sedimenting at 10-20s is clearly depleted in histones H2A and H2B. In addition to the nascent histones H3 and H4, one other protein is consistently found associated with the newly replicated chromatin. This 20,000 dalton protein (labeled X in Figures 4 and 5) does

not accumulate like the histones but turns over with a half-life of 30 min (results not shown). Together with the histones, the 20,000 dalton protein is the most abundant nascent protein in the nucleus after a short pulse with 35S-methionine (see Figures 4b, 5a and 5b, fraction P) or 3H-lysine (results not shown), although no protein can be detected at that position on the stained gels (Figure 4a, fraction P). Evidence for the association of the 20,000 dalton protein with nascent chromatin stems from the fact that after short 2 min pulses with labeled amino acids, the protein is found in 10-15s structures and after longer pulses it is found in 10-30s structures, a distribution which parallels the distribution of the nascent H3 and H4 histones and the maturation of the nascent chromatin described above. A slightly smaller radioactive protein present in the soluble nucleoplasm (X’ in fraction 1 of Figures 4b, 5a and 5b) could represent a cleavage product of X. Preliminary fingerprinting analysis by proteolysis in SDS (Cleveland et al., 1977) suggests that the 20,000 dalton protein is distinct from the histones. Further characterization of this protein is hampered by its high turnover and its.extremely low intracellular concentration. Discussion Our results indicate that the newly made histones are not deposited on bulk chromatin but associate instead with the newly made DNA. Cremisi, Chestier and Yaniv (1977) have recently reached the same conclusion in their analysis of the fractionated nascent SV40 minichromosome. Thus the random association of new histones with cellular chromatin observed by other investigators (Jackson et al., 1975, 1976; Seale, 1976; Hancock, 1978) must probably be due to protein rearrangements induced by the in vivo DNA density labeling and/or the in vitro formaldehyde cross-linking of chromatin. The new histones are not deposited synchronously on the new DNA. Nascent histones H3 and H4 appear to be deposited first on the newly made DNA, a result which is consistent with the fundamental role of H3 and H4 in nucleosome organization (Cammerini-Otero, Sollner-Webb and Felsenfeld, 1976; Sollner-Webb, Camerini-Otero and Felsenfeld, 1976; Bina-Stein and Simpson, 1977; Oudet et al., 1978; Wilhelm et al., 1978). Nascent histones H2A and H2B seem to be deposited 2-10 min after the deposition of the nascent H3-H4 histones, and nascent histone Hl lo-20 min later. In the case of Hl, our results cannot unambiguously distinguish between random association to bulk chromatin as opposed to late association to nascent chromatin, since both nascent Hl in chro-

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FRACTION NUMBER Supranucleosomal

Structures

Cells were’libeled for 2 min with 1 mCi of ‘%-methionine. 2 x IO’ nuclei were incubated with 80 U of staphylococcal nuclease for 15 min on ice and processed as described in the legend to Figure 2. Protein in the soluble sucrose gradient fractions (-20% of the total chromatin) and in the insoluble pellet (P; -80% of the total chromatin) was extracted and analyzed as described in Experimental Procedures. (a) Coomassie blue-stained SDS gel. The protein in fractions 1 and 2, which runs slightly slower than histone H3. is staphylococcal nuclease. (b) Fluorograph of the same gel. The intense labeling of H3 in this figure and in Figures 5a and 5b reflects its higher methionine content (histone H3 has two methionine residues per mole, while histone H4 contains only one; Alfageme et al., 1974).

matin and total Hl in bulk chromatin peak at about 50s in our gradients. We favor the latter possibility, however, because of the well known role of HI in chromatin compaction (Finch and Klug, 1976; Nell and Kornberg, 1977; Renz et al., 1977). Indeed, the nascent chromatin would compact as soon as the nascent Hl binds to it, and thus lo-20 min after replication, both nascent and bulk chromatin would display a similar condensed state, as observed . The rate of movement of each replication fork is about 3 kb/min in the Drosophila tissue culture cells (Blumenthal, Kriegstein and Hogness, 1973). Thus the observed 2-10 min gap between deposition of H3-H4 and deposition of H2A-H2B would imply that the first 30-150 new nucleosomes behind the fork may not contain histones H2A-H2B. McKnight and Miller (1977) have observed nucleosome structures immediately behind replicating forks in Drosophila embryos, but H3-H4 nucleo-

somes cannot be distinguished on the electron microscope from nucleosomes carrying the full histone complement (Wilhelm et al., 1978). More recently, McKnight, Bustin and Miller (1978) have reported that those nucleosomes behind the fork react with both anti-H3 and anti-H2B immunoglobulins. Such a result would be in disagreement with the data presented here, although it is possible that the mechanism of histone deposition is differentthat is, faster-in the Drosophila embryo. The sincitial blastoderm embryos used by McKnight et al. (1978) have an unusually short 3 min S phase (Rabinowitz, 1941) and a large pool of all the required histones (D. Brutlag, personal communication). On the other hand, our Drosophila tissue culture cells have an S phase of about 8 hr and show no appreciable histone pools. Indeed, the tight coupling between histone and DNA synthesis observed in tissue culture cells (Weintraub and Holtzer, 1972; Housman and Huberman, 1975;

Assembly 975

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in the Solubilized

Chromatin

Cells were labeled for 10 min with 1mCi of YS-methionine. nuclease for 15 min on ice and processed as described gradients are shown in this figure.

4 5 6 7 8 9 10 1112 13 P

FRACTION as a Function

of Nuclease

Procedures

Cells and Growth Conditions Drosophila melanogaster tissue culture cells (GM3 line; Mosna and Dolfini. 1972) were grown in suspension in 100-1000 ml Wheaton spinner flasks at 26°C in Schneider’s medium (Schneider, 1966) with 10% heat-inactivated fetal calf serum (FCS; Gibco). The cell concentration was kept at 0.5-l .5 x 1O’cells per ml by daily dilutions with fresh media. Cell doubling time was about 20 hr. Radioactive Labeling of the Cells For DNA labeling experiments, 10 #Zi of ‘+C-thymidine (NEN; 50 mCi/mmole) were added to the culture for 24 hr. and then 1 mCi of methyl-3H-thymidine was added for 20 sec. Cell growth was stopped by the addition of a 5 fold excess of ice-cold Schneider’s medium without FCS. and the cells were immediately pelleted at 3000 rpm for 5 min in the cold. For the pulse-chase experiment shown in Figure 3, the cells were resuspended in Schneider’s medium with FCS and 3 mM thymidine and grown for an addi-

NUMBER

Concentration

2 x 10’ nuclei were incubated in the legend to Figure 4. Only

Searle and Simpson, 1975) may not exist in the early rapidly dividing nuclei of the Drosophila embryo. Our results are not in disagreement with the finding that the new histones assemble and segregate as a conserved H3-H4-H2A-H2B octamer (Leffak, Grainger, and Weintraub, 1977); the long labeling times used in the experiments of Leffak et al. (1977) could not have detected the 2-10 min lag between the deposition of histones H3-H4 and histones H2A-H2B. Experimental

-H28 lH2A lH4

with (a) 30 U or (b) 120 U of staphylococcal the fluorographs of the fractionated sucrose

tiona12 and 10 min at 26°C. For protein labeling experiments, the cells were grown in medium without methionine (-met) for lo-12 days. The cells grew with the same doubling time (20 hr) for at least 15-20 days in the -met medium. The partially methionine-starved cells were labeled with 1 mCi of 3BS-L-methionine (NEN; 400 Ci/mmole) for various times and pelleted as before. Nuclei Pnparatbn Pelleted cells from each 100 ml of culture were resuspended in 25 ml of ice-cold solution I [0.005 M PIPES (pH 7.0), 0.065 M KCI, 5/ 5% (w/v) sucrose], and the cells were lysed by the addition of 25 ml of solution II (solution I + 0.2% Nonidet P40). The solutions were mixed and the nuclei were pelleted at 3000 rpm for 5 min. The pelleted nuclei were resuspended by brief vortexing in 50 ml of solution Ill (solution I + 0.1% Nonidet P40). The nuclei were washed 4 times in solution Ill by successive resuspension and sedimentation as before. The four washings completely eliminate contaminating ribosomes (both ribosomesand supranucleosomal structures have similar S values and thus appear on the same fractions in the isokinetic sucrose gradients). Nuclease Digestion The final nuclear pellet was resuspended in 1.5 ml of solution IV (solution I + 1 mM CaCI,) at lOa nuclei per ml or less. The nuclear suspension was split into six 0.25 ml aliquots, and varying amounts of staphylococcal nuclease (Boehringer) were added to each aliquot (20-400 U). Incubation was usually for 15 min on ice. Reaction was stopped by the addition of 0.25 ml of a solution containing 0.005 M PIPES (pH 7.0), 0.065 M KCI, 10 mM EGTA. The nuclear suspension was gently mixed, and after a further 10 min on ice, the clear mixture was spun at 1000 rpm for 5 min to pellet the nuclei and insoluble debris. Normal nuclear morphology was maintained throughout this treatment as monitored .by

Cell 976

both light and electron microscopy. PIPES buffer (a nonmetal ion-binding buffer; Good et al., 1966) and EGTA (a specific Ca++ chelator) are essential for keeping the chromatin in its native condensed state; both Tris buffer and EDTA unravel our supranucleosomal structures (M. L. Wong and A. Worcel, unpublished observations).

Fractionatkn

of the Dlgested

Chromatin

biomacromolecules.

J. Ultrastructural

Res. 35, 147-167.

Felsenfeld. G. (1976). Chromatin. Nature277, 115-122. Finch, J. T. and Klug, A. (1976). Solenoidal model for superstructure in chromatin. Proc. Nat. Acad. Sci. USA 73, 1697-1901. Finch, J. T.. Nell, M. and microscopy of defined lengths USA 72, 3320-3322.

Kornberg, R. D. (1975). Electron of chromatin. Proc. Nat. Acad. Sci.

The supernatant from the low speed spin, containing the soluble chromatin fragments which leaked out of the nuclei (usually IO50% of the total “H-thymidine radioactivity, depending upon the extent of digestion), was layered on top of isokinetic sucrose gradients containing 0.005 M PIPES (pH 7.0), 0.100 M KCI, 0.2 mM EGTA (C = 5%, particle density 1.51; McCarty, Vollmer and McCarty, 1974). Centrifugation was in a Beckman rotor SW41 for 3.5 hr at 41 ,OOtT rpm at 4°C. The gradients were fractionated in a cold room and absorbance at 260 rnp was monitored in a flowthrough cell (ISCO). Radioactivity measurements and SDS-polyactylamide gel electrophoresis were performed as previously described (Benyajati and Worcel, 1977). Theentire original nuclear suspension, including the soluble nucleoplasm and the insoluble nuclear pellet, was recovered and displayed on the gels.

Freedlander. F. E., Taichman, L. and Smithies, 0. (1977). Nonrandom distribution of chromosomal proteins during cell replication. Biochemistry 16, 1602-1606.

Housman, D. and Huberman. J. (1975). DNA replication fork movement during cells. J. Mol. Biol. 94, 173-161.

Changes S phase

Electron

Hozier, J., Renr, M. and Nehls, S. (1977). evidence for an ordered superstructure mosoma 62, 301-317.

The chromosome of nucleosomes.

Jackson, V.. Granner, D. K. and Chalkley. histones onto replicating chromosomes. USA 72, 4440-4444.

R. (1975). Deposition of Proc. Nat. Acad. Sci.

Mkroacopy

Samples from individual fractions of the sucrose gradients were applied to glowed carbon-coated grids (Dubochet et al., 1971) which were then stained with a dilute solution of uranyl acetate (0.01% in ethanol). The grids were dried under a heat lamp and rotary-shadowed with platinum at an angle of 6”. Micrographs were taken using a Philips 300 electron microscope.

Acknowledgments This research has been supported by grants from the USPHS and the American Cancer Society. A. W. is the recipient of a USPHS Career Development Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. Received

July 27,1976;

revised

September

1,1976

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Good, N. E., Winget, G. D., Winter, W., Connolly. and Singh, M. M. (1966). Hydrogen ion buffers research. Biochemistry5, 467-477. Hancock, R. (1976). DNA into chromatin.

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Hildebrand. C. E. and Walters, R. A. (1976). Rapid assembly of newly synthesized DNA into chromatin subunits prior to joining the small DNA replication intermediates. Biochem. Biophys. Res. Commun. 73, 157-163. in the rate of in mammalian fiber: Chro-

Jackson, V., Granner, D. and Chalkley, R. (1976). Deposition of histone onto replicating chromosomes: newly synthesized histone is not found near the replication fork. Proc. Nat. Acad. Sci. USA 73, 2266-2269. Kornberg. R. (1977). 46, 931-954.

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McCarty, K. S., Vollmer, R. T. and McCarty, K. (1974). Improved computer program data for the resolution and fractionation of macromolecules by isokinetic sucrose density gradient sedimentation. Anal. Biochem. 61, 165-163. McKnight, S. L. and Miller, 0. L., Jr. (1977). Electron microscopic analysis of chromatin replication in the cellular blastoderm Drosophila melanogaster embryo. Cell 72, 795-864. McKnight, S. L., Bustin. M. and Miller, 0. L. (1976). Electron microscopic analysis of chromosome metabolism in the Drosophila melanogasfer embryo. Cold Spring Harbor Symp. Quant. Biol. 42, 741-754. Mosna, G. and Dolfini. somal characterization Drosophila melanogaster.

S. (1972). Morphological of three new continuous Chromosoma 38, l-9.

Blummenthal. A. B., Kriegstein, H. J. and Hogness, D. S. (1973). The units of DNA replication in Drosophila melanogaster chromosomes. Cold Spring Harbor Symp. Quant. Biol. 38, 205-223.

Nell, M. and Kornberg, nuclease on chromatin Biol. 189, 393-404.

R. D. (1977). and the location

Camerini-Otero, R. D., Sollner-Webb, B. and Felsenfeld, G. (1976). The organization of histones and DNA in chromatin: evidence for an arginine-rich histone kernel. Cell 8, 333-347.

Oudet, P., Germond, Chambon. P. (1976). Sot. B 283, 241-256.

Cleveland, D. W., Fisher, S. G.. Kirschner, M. W. and Laemmli, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate by gel electrophoresis. J. Biol. Chem. 252, 11021106.

Rabinowitz, M. (1941). Studies on the cytology and early embryology of the egg Drosophila melanogaster. J. Morphol. 69, l-49.

Bina-Stein, contraction

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R. T. (1977). Specific folding H3 and H4. Cell 77, 609-616.

Cremisi. C., Chestier, A. and Yaniv, ciation of newly synthesized histones Cell 12, 947-951. Dubochet, J.. Ducommon, A new preparation method

and

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of larval eye-antenna1 disks and cultured in vitro. J. Embryol. Exp.

Scale,

relationships

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Temporal

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protein

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synthesis, tein. Proc.

of Newly

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