THE .JOURNAL OF BIOI.OGICAL CHEMISTRY Vol. 25i, No. 14, … · 2001-08-25 · Vol. 25i, No. 14,...

THE .JOURNAL OF BIOI.OGICAL CHEMISTRY

Printed tn U S A. Vol. 2 5 i , No. 14, Issue of duly 25, pp. 85074515, 1982

Association of Newly Synthesized Histones with Replicating and Nonreplicating Regions of Chromatin*

(Received for publication, October 28, 1981)

Anthony T. Annunziato$, Rudolf K. Schindler, Michael G. Riggs, and Ronald L. Seales From the Department of Cellular Biology, Scripps Clinic and Research Foundation, La Jolla, California 92037

Histone deposition in HeLa cells has been studied by monitoring the fractionation and electrophoretic mobility of pulse-labeled histones under conditions that separate newly replicated from bulk chromatin DNA. The separation efficiency of these two methods is -70%. Following micrococcal nuclease digestion, chromatin was fractionated by salt elution. 50-65% of the newly synthesized histones eluted with bulk chromatin at NaCl concentrations between 0.1 and 0.3 M and were further shown to co-electrophorese with bulk chromatin DNA, not with the more extensively digested newly replicated chromatin DNA contained in those fractions.

The remaining chromatin fractions, solubilized with 0.4-0.6 M NaC1, were several-fold enriched in nascent DNA (Annunziato, A. T., Schindler, R. K., Thomas, C. A., Jr., and Seale, R. L. (1981) J. Biol. Chem. 256, 11880-11886) and were correspondingly enriched for the balance (35-50%) of newly synthesized core histones. This fraction of newly synthesized core histone may be preferentially deposited onto newly replicated DNA.

In contrast, histone H1 showed little tendency toward deposition onto new DNA. Within 15 min all new core histones attained the same solubility and electrophoretic mobility as bulk chromatin.

We conclude that newly synthesized histones are deposited onto both replicating and nonreplicating regions of chromatin.

In conceptualizing the replication of a linear series of nucleosomes, it is clear that, even with complete retention of the histone octamers preceding the replication fork, the progeny DNA duplexes will be 50% deficient in histone protein until additional histones become complexed with the new DNA. Several studies on the structure of the DNA component of chromatin synthesized either with or without concurrent protein synthesis have confi ied this prediction and have indicated that, due to the putative inheritance of pre-fork nucleosomes, approximately half of newly replicated DNA is nucleosomal, and the other half is non-nucleosomal (7,30-32,4453, 54)’; when protein synthesis is allowed to proceed, the non- nucleosomal component is quickly assembled into nucleosomes (44).

The dual structural characteristics of the new (chromatin)

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

8 Recipient of National Research Services Award Gm 07309. 8 Recipient of National Institutes of Health Grants GM27950 and

‘ A . T. Annunziato and R. L. Seale, manuscript submitted for AG 02305.

publication.

DNA are correspondingly reflected in its dual physical and biochemical properties. Nuclease-fragmented, newly replicated chromatin has recently been demonstrated to have bimodal solubility characteristics when subjected to elution by stepwise increments in ionic strength (44). In this fractionation procedure, 50-70% of newly replicated DNA is co-eluted with bulk chromatin DNA at low ionic strength, and has a nucleosomal configuration. In contrast, 30-50% of new DNA is quite refractory to solubilization, requiring exposure to 0.4-0.6 M NaCl for release, and this DNA is not organized into nucleosomes (44). Thus, the salt elution procedure appears to achieve a biochemical separation of the two opposite arms of the replication fork, which have been shown to correspond to the lagging (non-nucleosomal) strand and the leading (nucleosomal) strands in SV40 (7).

Although the DNA component of nascent chromatin shows bimodal properties, confii ing intuitive predictions, by the same reasoning nascent core histones are not expected to show comparable dual characteristics. If the lagging strand were the site of deposition of all new core histones, then the majority of these proteins should show properties identical to that of the nascent lagging strand chromatin.

Attempts to determine the deposition site for new histones have yielded a complex and contradictory literature (reviewed in Refs. 1-3). It has been suggested that the newly synthesized histones are deposited onto new DNA (4-7), old DNA (8-11), and both new and old DNA (12-14,55). Such diverse conclusions concerning the site of nucleosome assembly might stem from differences inherent to the cell types studied or from variations in experimental procedures.

It is generally assumed that the mechanisms of somatic cell replication have been conserved among higher organisms. On the other hand, the influence of experimental protocol is an important consideration, since newly synthesized histones may require several minutes to become assembled into stable nucleosomes (6,13,43,55). New histone-DNA complexes may have a structure very different from that of mature chromatin, as evidenced by the dissociation of a fraction of newly synthesized nucleosomal histones by 0.45 M NaCl, and by their selective sensitivity to digestion with trypsin (43). Moreover, in using micrococcal nuclease to solubilize chromatin, a sig- nificantly enriched fraction of nascent DNA may escape analysis, due to its insolubility, as a result of its possible association with insoluble nuclear structures (e.g. the nuclear matrix) (15-17, 44). Such considerations stress the importance of examining 100% of the new histones and DNA in the nucleus when studying histone deposition.

Because we have applied two complementary fractionation procedures that effectively distinguish nascent chromatin from bulk chromatin, we are in a unique position to pose the question of whether or not newly synthesized core histones are exclusively deposited onto nascent DNA. Evidence is presented which is consistent with the bimodal deposition of

8507

8508 Deposition of Newly Synthesized Histones

new core histones onto both replicating and nonreplicating regions of chromatin. The bimodal deposition may be the source of previous conflicts in the literature on this subject.

MATERIALS AND METHODS

Cell Culture and Labeling-HeLa cells were maintained in expo- nential growth in spinner culture at 37 "C in Eagle's minimal essential medium supplemented with 5% calf serum.

For long term labeling of histones, cells were incubated for 20-40 h with either 0.06 pCi/ml each of [I4C]arginine and ["Cllysine or 5.5 pCi/ml of [3H]arginine and [3H]lysine in culture medium depleted 80% in those amino acids.

For amino acid pulse-labeling, cells (-4 X lo5 cells/ml) were concentrated 20-fold (2-min pulse) or %fold (15-min pulse) in pre- warmed arginine- and lysine-free medium and allowed to equilibrate for 10-20 min at 37 "C. The cells were harvested, resuspended at the same concentration in arginine- and lysine-free medium, allowed to equilibrate for an additional 5-10 min, and incubated for 2 min with 50-60 pCi/ml of [3H]lysine (60 Ci/mmol) and arginine (30 Ci/mmol). In control experiments, the incubation of cells in arginine- and lysine- free medium did not alter the rate of total protein synthesis for over 1 h. 15-min labeling was performed at 5 pCi/ml. Labeling was halted by diluting cells into ice-cold CB buffer (1 mM Tris, 25 mM KC1, 0.9 mM MgC12, 0.9 mM CaCI2, 0.14 r n ~ spermidine, 1 mM Na-butyrate, pH 7.6) containing 0.1% sodium azide.

Nuclear Isolation, Digestion with Micrococcal Nuclease, and Chromatin Fractionation-Cells were washed twice in CB buffer; nuclei were then isolated, resuspended in CB buffer a t a concentration of 46 A2m/ml (measured in 0.3 M NaOH), and digested with micrococcal nuclease as described (44). In most cases nuclei were digested for 2 min at 37 "C with 1.2 units of micrococcal nuclease (Sigma)/ml. The reaction was terminated by the addition of 100 mM ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (pH 7.6) to a final concentration of I m, and cooling to 4 "C.

Following nuclease digestion, nuclei were gently pelleted (1400 X g for 10 min) to yield a supernatant fraction (SO) containing acid- soluble nucleotides and soluble proteins. Chromatin was then fractionated according to the method of Sanders (18) by resuspending the nuclear pellet, sequentially, in CB buffer containing 0.5 mM phenylmethylsulfonyl fluoride and 1 mM ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid, plus 0.1,0.2,0.3,0.4, or 0.6 M NaC1. At each salt concentration the nuclei were pelleted (1400 X g ; 10 min) and the supernatant fractions, denoted S.1, S2, S.3, S.4, and S.6, respectively, were collected. A total soluble chromatin fraction, denoted T, was produced by lysing nuclei with 2 mM EDTA, pH 7.2, immediately after digestion, and removing the insoluble material by centrifugation (12,800 X g for 10 rnin). A second fraction, denoted T.2, was similarly obtained by nuclear lysis after the 0.2 M NaCl elution step.

In experiments performed in the absence of salt, pulse-labeling of cells was halted with 5 volumes of ice-cold Buffer A (3 m~ MgCL, 5 mM Tris, pH 7.6, M sodium tetrathionate), containing 0.1% sodium azide, and nuclei were isolated in buffer A. For micrococcal nuclease digestion, CaC12 was added to 2.5 X M. Digestion was performed at 37 "C for 2 min at 0.1 unitlA2W unit chromatin (measured in 1% SDS') and was terminated by the addition of ethylene glycol bis(b-aminoethy1 ether)-N,N,N',N'-tetraacetic acid to 1 mM. Nuclei were pelleted by centrifugation in an Eppendorf microfuge for 5 min, and the SI supernatant was removed. The nuclear pellet was dispersed in 0.2 mM EDTA, pH 7.6, for 12 h at 4 "C prior to centrifugation to separate the SI1 supernatant from the final pellet, P. The yields in these fractions are described in the text.

Chromatin was stripped of histone H1 and non-histone proteins by washing with 0.45 M NaCl (43). Briefly, the SI1 and the P fractions were adjusted to 0.45 M NaCl (insoluble material in the pellet fraction was removed by a 5-min spin in an Eppendorf microfuge), and the supernatants were added to 5 ml of stripping buffer (0.45 M NaCl, 5 m Tris, pH 7.4, 1 mM EDTA, 2 mM mercaptoethanol), layered onto 2 ml of 50% (w/w) sucrose in this buffer, and spun at 45,000 rpm for 65 h in the Beckman 50Ti rotor at 4 "C. The pellet of nucleosomes, free of histone HI and non-histone proteins, was dissolved in 0.2 mM EDTA for further manipulations.

Histone Extraction and Gel Electrophoresis-Histones were extracted from the chromatin fractions with 0.4 M HrS04 for 12 h,

The abbreviations used are: SDS, sodium dodecyl sulfate; DNP, deoxyribonucleoprotein.

precipitated with 25% trichloroacetic acid, washed with acetone, and dissolved in sample buffer. Alternatively, the chromatin fractions were ethanol-precipitated, dried, and solubilized in sample buffer. Electrophoresis of proteins in SDS-polyacrylamide gels was performed according to the method of Thomas and Kornberg (19). Analysis of deoxyribonucleoprotein particles in agarose-polyacrylamide (DNP) gels was as described (44,46). DNP gels were prepared for fluorography using EN3HANCE (New England Nuclear); SDS- polyacrylamide gels were impregnated with 2,5-diphenyloxazole (20, 21). Gels and films were scanned in a Joyce Loebl and Co. densitom- eter.

Radioactivity Determinations-Following electrophoresis, proteins were stained with Coomassie blue; duplicate samples of individual histones were excised, solubilized with 0.2 ml of NCS (Amersham Corp.), and counted in 4 g/liter of 2,5-diphenyloxazole-toluene. Chro- matin samples were precipitated together with 50 pg each of DNA and albumin onto glass fiber filters (Reeve Angel 934-AH, Whatman) with 10% trichloroacetic acid, washed twice with 10% trichloroacetic acid, and twice with 95% ethanol, solubilized with NCS (Amersham Corp.), and counted. (The fraction of I4C counts/min in the 3H channel was corrected by the external standard technique.)

RESULTS

Salt Elut ion of Newly Synthesized Histones-When nuclease-digested nuclei are maintained in the presence of mag- nesium ions, chromatin can be fractionated by incrementally raising the ionic strength of the medium with NaCl (see "Materials and Methods"), thereby solubilizing chromatin fragments of progressively higher molecular weight (18,22,44; see also Fig. 3A). As previously shown (44) the elution of newly replicated chromatin DNA does not parallel that of bulk chromatin. Rather, a significant fraction of new chromatin DNA is insoluble at the low to moderate ionic strengths (0.1-0.3 M) at which bulk chromatin is released, rendering the chromatin fractions solubilized at higher ionic strengths (0.4-0.6 M) enriched in new DNA. To determine whether newly synthesized histones were similarly resistant to salt elution, nuclei were isolated from HeLa cells that had been incubated with r3H]arginine and ['Hllysine either for 2 min, or for one generation, and chromatin was fractionated. The nuclear proteins released at each salt concentration were then subjected to electrophoresis in SDS-polyacrylamide gels, and analyzed by staining and by fluorography. As will be shown later, different amounts of chromatin (and therefore of histones) were solubilized at each step in the fractionation, with greater than 80% of the bulk histones released prior t o the 0.4 M NaCl extraction. Samples were therefore normalized for protein insofar as possible, given the low yield of bulk histones in the S.4, S.6, and P fractions (Fig. 1, A and B).

The positions of the labeled proteins are presented in the gel fluorographs (Fig. 1, C and D). While the radioactivity of the histones labeled for one generation (Fig. IC) parallels the mass distribution of the stained histones (Fig. lA), core histones labeled for 2 min are strongly enriched in the S.6 and P fractions (Fig. 1, B and D) . This is most readily observed for H4, although all four core histones are enriched in the high salt eluates. Histones H3 and H4 are under-represented in Fig. 1D relative to their uniform labeling pattern due to 1) a lag in the deposition of H3 and H4 relative to the rapid assembly of H2A and H2B (43) and 2) the rather facile nature of the initial association of the new H3 and H4 with chromatin, resulting in their preferential loss (13, 55). The net result is that the fractions which were previously identified as being enriched in newly replicated DNA show enrichment for newly synthesized core histones.

The results of Fig. 1 are consistent with data that has been interpreted to indicate assembly of new histones with new DNA (5, 6, 13, 14); however, analysis of this result by inspec- tion of fluorographs is problematic for several reasons. First, a comparison of radioactivity with staining intensity reflects

Deposition of Newly Synthesized Histones 8509

2 0 HR A 0 .I .2 .3 4 .6 P - ."

H I =

H 3 L H 2 A . B H 4-

c 0 I .2 .3 .4 .6 P

H I

2 MIN 0 I .2 3.4.6 P R

0 .I 2 3.4 6 P D

. .- = ":

FIG. 1. Salt elution of new and old histones. HeLa cells were labeled for 20 h ( A and C) or 2 min ( B and D) with ['Hlarginine and ["Hllysine. Nuclei were digested with micrococcal nuclease, and chromatin was fractionated by stepwise elution with NaCl (see "Materials and Methods"). Total proteins from each fraction were subjected to electrophoresis in SDS-polyacrylamide gels; the gels were stained (A and B ) and analyzed by fluorography (Cand D). Lanes 0-Gcorrespond to the NaCl molarity used to elute each fraction. Lane P contains proteins from the final pellet fraction. Arrowheads ( B and C): non- histone proteins bracketing HIA and HIB.

specific activity, not absolute amount. For example, the 0.3 M NaCl fraction contains the most radioactivity (shown be- low), yet it appears depleted in the fluorograph (lane .3, Fig. 10). Thus, variations in the quantity of sample loaded onto the gel should be noted. Second, subtle differences in the elution of some proteins can easily be overlooked. By inspec- tion one might conclude that the elution of new H1 correlated with that of the new core histones; it will be shown by quantitation that this is not the case. Third, the co-elution of new proteins and DNA does not prove their association into the same structures. For example, histone H1 is dissociated from chromatin by 0.4-0.6 M NaCl, and is released from nuclei in the absence of nuclease digestion (23, 24). Moreover, we later show that for a substantia1 fraction of chromatin, the new histones are not associated with new DNA, although they co-purify into the same fractions.

Quantitation of the Enrichment for New Histones-To determine the percentage of both new and old histones released into each of the salt eluates, cells were incubated for 40 h with ['*C]arginine and [14C]lysine, and then for 2 min with ["Hlarginine and ["Hllysine. Acid-extracted nuclear proteins were subjected to electrophoresis in SDS-polyacrylamide gels, stained histone bands were excised from the gel, and the proteins were solubilized and counted. Fig. 2B shows the elution profiies for new uersus old H3 and H4, and Fig. 2 0 t h e data for H2A and H2B.

In all cases, the largest fraction of the old core histones (35~45%) was eluted with 0.3 M NaC1, with over 90% released into the first four fractions (S.1-S.4). In contrast, 20-30% of the new histones were insoluble in 0.4 M NaCl, with the result that the S.6 and P fractions were relatively enriched for these

proteins. The relative enrichment of newly synthesized core histones (expressed as per cent "/per cent I4C) is shown in Fig. 2, A and C; the pellet fraction (P) is 5- to 10-fold enriched in new core histone relative to the 0.3 M NaCl eluate. Thus, the elution of newly synthesized histones correlates with that of new DNA (44).

The amounts of histones H3 and H4 that co-purify with nascent DNA predominate over those of H2A and H2B, in accord with results of others (6, 12-14, 33, 55). Although this preponderance is consistently observed, it is less than found in cells treated with metabolic inhibitors (13).

Unlike the core histones, the newly synthesized H1 histones (Fig. 2, E and F ) showed little tendency toward enrichment in the S.4, S.6, and P fractions (Note: The 0.1 M NaCl eluate lacks H1 (Fig. 1; see also Refs. 22 and 25). Similarly, H1 was typically absent from the pellet fraction after the 0.6 M NaCl elution step, although in some cases a trace (less than 3%) was retained (Fig. 2F). The strongly labeled proteins present in

0

' 6 U

T $ 4 z

2

* I I I I

E 40

2 30 W z 2 0

10 -_ 2 .3 .4 6 P

CONCENTRATION NOCIIY)

FIG. 2. Elution profiles of new and bulk core histones (A-D), co-elution of new and old H1 (E and F ), and maturation of new histones (G and H ) . Cells were prelabeled for 40 h with ["'Clarginine and ["Hllysine, and then incubated for 2 min with ['Hlarginine and [:'H]lysine. Isolated nuclei were digested with 1.2 units of micrococcal nuclease/ml digestion buffer and chromatin was fractionated as in Fig. 1. Acid-extracted proteins from each eluate (0-6) and the final pellet (P) were subjected to electrophoresis in SDS-polyacrylamide gels. Stained histones were excised, solubilized, and counted. Hadio- activity is expressed as per cent of total. B, elution profiles of old H3 (O), new H3 (O), old H4 (A), and new H4 (A). D, elution profiles of old H2A (O), new H2A (01, old H2B (A), and new H2B (A). The relative enrichment for new histones is expressed as (per cent .'H counts/min)/(per cent I4C counts/min) for each fraction: A, V, H3; 0, H4. C, W, H2A 0, H2B. E and F, H I histones were labeled, extracted, and analyzed as in A-D. F, average value of old (0) and new (0) HIA and HIB. E , per cent "H/per cent "C. G and H , cells were prelabeled for 40 h with ["Clarginine and ["Hllysine and then incubated for 15 min with [:'H]arginine and ['Hllysine. Acid-extracted core histones were analyzed as in A-D. H, average value of old (0) and new (0) H2A, H2B, H3, and H4. G, per cent "H/per cent ''C.


the pellet lane in Fig. 1D are not H1 histones, but closely migrating non-histone proteins at the upper and lower bound- aries of the two H1 species, as shown in the stained gel (B) and indicated by arrowheads (lane P, Fig. 1, B and D)). Despite the solubility of the H1 histones in 0.4-0.6 M NaCl, it is clear that, unlike new core histones, newly synthesized H1 is neither depleted in the S.2 and S.3 fractions, nor enriched in fractions insoluble in 0.3 M NaC1. The striking difference between elution profiies of newly synthesized H1 and core histones suggests that H1 is deposited onto chromatin at sites different from those of core histone assembly, in accord with other reports (13, 14,55).

Maturation of Nascent Chromatin-Within 10 min, the differential elution pattern of new uemus bulk chromatin DNA is no longer observed, and the ratio of new/old chromatin DNA is constant for each step in the fractionation procedure (44). In order to obtain further information regard- ing the putative association of the salt-insoluble histones with new DNA, the time required for the maturation of new histones onto salt-soluble complexes was examined. Histones were therefore prelabeled for 40 h with ['4C]arginine and ["C] lysine, and then labeled for 15 min with [3H]arginine and [3H] lysine. Acid-extracted core histones were then analyzed as in Fig. 2, B and D. Only 2-5% of the new histones were retained in the S.6 and P fractions when the incubation period was extended to 15 min (Fig. 2H) and the elution patterns of new and old core histones were nearly superimposable. Although the 3H/14C ratios were elevated in the high salt eluates after 15 min of labeling, this is probably an overestimate due to a combination of factors, including a lag in the assembly of new histones onto chromatin (43, 55), pool effects (26, 37, 43), the use of continuous 15-min label rather than a true "chase," and statistical variations as the 3H counts/min approach back- ground levels. Within 15 min most new histones are "chased" into a conformation possessing the same solubility as bulk chromatin.

Distribution of Newly Synthesized DNA and Histones Sol- ubilized in Low Salt Buffer-To ensure that the results of the preceding experiments were not salt-induced redistribution artifacts, a similar set of experiments was performed on nuclei that were never exposed to ionic strengths that could induce core histone redistribution. Cell fractionation and nuclease cleavage were performed at low ionic strength (3 mM MgC12, 10 mM Tris, pH 7.6, and 2.5 X M CaC12) and chromatin was solubilized and maintained in 0.2 m EDTA.

Using the protocol of Todd and Garrard (46), three fractions of digested chromatin were obtained (Table I). The fist supernatant, SI, obtained after sedimenting the digested nuclei, contained approximately 10% of total acid-insoluble DNA, and consisted of monomeric nucleosomes. The second supernatant, SII, obtained by dispersion of the digested nuclear chromatin in 0.2 m EDTA, contained about 80% of the acid-insoluble bulk DNA as mono- and polynucleosomes. The pellet, P, contained 10% of bulk DNA as insoluble higher molecular weight polynucleosomal chromatin, plus other insoluble elements including the nuclear membrane and matrix.

The distribution of newly synthesized DNA (Table IA) shows 2-fold enrichment in the insoluble pellet, relative to bulk chromatin, in correspondence with results of others (15-17, 34,44).

In order to determine the distribution of new histones in the SI1 and P fractions, cells were labeled for one generation with [14C]lysine and then for 2.5 min with [3H]lysine. The SI1 and P fractions were acid-extracted, and the acid-soluble proteins were separated in SDS-polyacrylamide gels; each band was excised and the amounts of 3H and 14C of each histone were determined (Table IB). On average, bulk core

TABLE I Distribution of total DNA, newly replicated DNA, and newly

synthesized histones in chromatin prepared in low ionic strength conditions

A. DNA

Fraction mldme, 18 midine, 2.5 R 3H/% P/SII %[';C]Thy- ' LSHIThy-

min

SI 10 12 1.2 SI1 81 70 0.86 P 9 18 2.00 2.33

B. Histone

H4 SI1 75 57 0.76 P 16 35

H3 2.19

SI1 2.88

71 57 P

0.8 17 37 2.18 2.73

H2A SI1 65 51 0.78 P 22 42 1.91 2.45

H2B SI1 74 60 P

0.81 17 36

H 1A 2.12

SI1 2.62

71 63 P 22 39

0.89 1.77

H1B 1.99

SI1 68 57 P

0.83 26 38 1.46 1.79

histone was distributed as 71% SI1 and 18% P, in contrast to newly synthesized core histones which were distributed as 56% SI1 and 37% P. The absolute values of bulk DNA and histone fluctuate, with the experiment, within the range of 70-80% (SII) and 10-20% (P); the important parameter is the degree of relative enrichment shown by the P/SII ratio. This represents a 2.6-fold enrichment in the P fraction, and corre- sponds to the %fold enrichment for new DNA (Table IA).

New histone H1 was distinctly less enriched in the insoluble chromatin, and, as will be demonstrated, the H1 deposition pattern does not follow that of the core histones.

Sites of Deposition of Newly Synthesized Histones onto Chromatin-Both newly replicated chromatin DNA (44) and the newly synthesized core histones (Fig. 2) were relatively depleted in low ionic strength eluates, and correspondingly enriched to similar degrees in high ionic strength fractions. Such correlations between DNA and histones in sedimenta- tion velocity and buoyant density gradients have been taken by many investigators to indicate their physical association (5, 6, 8-14, 33, 35). However, these correlations do not prove that these components are assembled into the same structures. In order to determine the distribution of new histones, we subjected the chromatin fractions in which there is a dramatic difference in distribution between bulk and newly replicated DNA (44) to electrophoresis in DNP gels.

HeLa cells were labeled for 30 s with ["]thymidine, or for 2 min with [3H]arginine and [3H]lysine, and nucleoprotein complexes from the chromatin fractions were subjected to electrophoresis under conditions which enable the separation of several distinct mononucleosome classes (44, 46, 48) (Fig. 3). A comparison of the ethidium bromide stain (Fig. 3A) with the fluorograph of newly replicated chromatin DNA (Fig. 3B) reveals that within 30 s newly synthesized DNA was complexed with all mononucleosomal species, as previously described (44), and that newly replicated chromatin DNA was digested to mononucleosomes more rapidly than bulk chromatin (6, 27-30, 44, 45). The greater sensitivity of nascent chromatin to micrococcal nuclease is particularly evident in the fractions which consist primarily of high molecular weight chromatin (ie. the T, S.3, and T.2 fractions (Fig. 3, A and B)). The T (total) and T.2 fractions were prepared as controls in

Deposition of Newly Synthesized Histones 851 1

A T 0 I .2 .3 4 .6 T 2

B T 0 .I .2 .3 .4 .6 T 2 ,.A .

F ’

... .

c T 0 .I .2 .3 .4 .6 T.2 -

FIG. 3. Association of newly synthesized proteins with bulk chromatin. Cells were labeled for 30 s with [:‘H]thymidine ( B ) or 2 min with [’Hlarginine and [SHIlysine (C); chromatin was fractionated and the fractions were subjected to electrophoresis in DNP gels (see “Materials and Methods”). Gels were stained with ethidium bromide ( A ) and prepared for fluorography ( B and C) . Lane designations correspond to the NaCl molarity used for elution. Lanes T and T.2 contain chromatin released from nuclei with 2 m~ EDTA without salt exposure (T), or after elution with 0.2 M NaCl (T.2) (see “Materials and Methods”). The positions of mono- (M) and dinucleosomes (D) are indicated. Note: Samples were normalized for DNA content for resolution in gels; yields of new and old chromatin are indicated in Fig. 2, and in Ref. 44.

which the exposure to salt has been eliminated (T) or reduced (T.2) in order to minimize the potential of salt-induced protein rearrangement.

The separation of newly synthesized from bulk DNA, by solubility, in the low and high ionic strength eluates is 3040% (44). In addition, the 30% of new chromatin DNA in the 0.3 M NaCl eluate can be almost completely separated from bulk chromatin electrophoretically (Fig. 3B). Thus, we estimate the level of discrimination of new from bulk DNA, as the sum of these two analyses, to be 70%.

The electrophoretic separation of new from old chromatin in the T, S.3, and T.2 fractions provides a direct means for determining the mode of histone deposition. If new histones are deposited preferentially onto nascent DNA at or near the replication fork, then the newly assembled DNA-histone com-

plexes should exhibit the sensitivity to micrococcal nuclease characteristic of nascent chromatin DNA (Fig. 3B and Refs. 6,27-30,34, and 44-45). However, when proteins were labeled for 2 min the labeling pattern clearly followed the distribution of bulk chromatin (Fig. 3C) and not that of newly replicated chromatin DNA (Fig. 3B). Mono- and dinucleosomes were also labeled, indicating possible association of new histones with the new DNA in these particles (Fig. 3C).

To make certain that the label associated with high molecular weight chromatin was in histone proteins, the lanes containing the T, S.3, and T.2 fractions were excised and subjected to electrophoresis in a second dimension in SDS- polyacrylamide gels. All three fractions gave equivalent results; the data for the total (T) are presented (Fig. 4). The bottom panel clearly shows that histones were the predomi- nant labeled species. A comparison of the distribution of the ethidium-stained, bulk chromatin (top) with the fluorograph (bottom) demonstrates that the distribution of new histones parallels that of bulk chromatin. No corresponding enrichment for newly synthesized histone was detected in the positions enriched in nascent chromatin DNA, i.e. the monomer and dimer (Fig. 3B, lane T).

The co-elution of protein radioactivity with bulk chromatin observed in the S.4 and S.6 eluates (Fig. 3) must be viewed with caution with regard to the distribution in DNP gels. At a salt concentration of 0.45 M, a fraction of newly deposited histone can be extracted from chromatin, and therefore may be subject to rearrangement (43). This has apparently had little effect on the elution profile of new core histones, since the pellet fraction (after salt extraction) remains enriched in these proteins (Figs. 1 and 2). Nevertheless, it is not possible at this time to assess the deposition site for new histones in the higher salt eluates (S.4, S.6, and P).

Stability of New Histone-DNA Complexes-One problem which must be addressed when analyzing protein-DNA inter- actions is the possible rearrangement of proteins during experimental manipulations. I t was therefore essential to test for redistribution of the newly synthesized histones during elution and electrophoresis. Taking advantage of the fact that the 0.1 M NaCl eluate consists almost exclusively of mono- and dinucleosomes (Fig. 3, lane . I ) , the following control

DNP - D M n n

SDS

1 e

= H I

H 3 JH2B ”’H2 A 7H 4

FIG. 4. Two-dimensional analysis of newly synthesized histones. Cells were labeled for 2 min with [,”H]arginine and [’Hllysine, and chromatin fractionated as in Fig. 2. Total EDTA-soluble chromatin (corresponding to lane T in Fig. 3) was separated in the fmt dimension in a DNI’ gel and stained with ethidium bromide (top); chromatin proteins were analyzed in the second dimension in a SDS- polyacrylamide gel and analyzed by fluorography (bottom). The positions of mono- (M) and dinucleosomes (D) are indicated


mixing experiment was performed. HeLa cells were labeled for 2 min with ["Hlarginine and ['Hllysine and the S.l fraction was prepared. A second fractionation series from unlabeled chromatin was prepared under identical conditions. The labeled 0.1 M NaCl eluate (monomers) was then mixed with each of the unlabeled high molecular weight chromatin fractions ( i e . T, S.3, and T.2) in proportions to approximate the amount of monomer present in total nuclease-solubilized chromatin (Fig. 3A), and subjected to electrophoresis. In the case of the S.3 fraction the samples were also mixed in the presence of 0.3 M NaCl and dialyzed against 2 mM EDTA. In all cases there was only minor rearrangement of total labeled proteins from the original monomers and dimers to high molecular weight chromatin (Fig. 5). Clearly, the labeling pattern of new histones observed in Figs. 3 and 4 cannot be explained by histone dissociation from (newly replicated) monomers. Iden- tical results were obtained for proteins labeled for one generation (not presented).

To verify that no exchange of core histones occurred in NaCl concentrations up to 0.3 M, the mixed S.l/S.3 sample (equivalent to lane c in Fig. 5) was analyzed in a second dimension polyacrylamide gel in the presence of SDS. No newly synthesized core histones exchanged with the histones of high molecular weight chromatin. A limited exchange of proteins was noted, accounting for the faint radioactivity in the upper portions of lanes c, e, g, and h in Fig. 5. By second dimension analysis, this was found to be entirely from non- histone proteins, and did not involve core histones. This corroborates the observation that, although new histones are more salt-labile than mature histones, the threshold for dissociation is 0.4-0.45 M NaCl (43).:'

Electrophoretic Behavior of Newly Synthesized Proteins in Chromatin-In order to examine more closely the association of newly synthesized histones on new versus mature DNA, two further approaches were taken. In one, whole nuclease-soluble chromatin containing pulse-labeled histones (some of which are incompletely assembled (43)), were examined by electrophoresis in DNP gels followed by electrophoresis in a second dimension SDS gel and densitometry.

Cells were incubated in ["Hllysine for 2.0 min; the SI1 fraction was separated in the first dimension as nucleosomes, and in the second dimension in SDS-polyacrylamide gels. The fluorograph of such a gel (Fig. 6) demonstrates several features of pulse-labeled proteins. The histones are displayed across the entire chromatin profde; the skew of the new core histones is opposite that of H1. Numerous non-histone proteins are also labeled, necessitating the resolution of individual histones from total chromatin proteins for analysis.

In order to quantitate the distribution of new core histones relative to bulk histone, scanning was performed along the long axis of individual histone protein bands, and scans from both the stained gel and the fluorograph were overlaid (Fig. 7). The scans were obtained from gels in which the chromatin was of greater average molecular weight than that shown in Fig. 6. Both radiolabeled H2A (H2B) ( A ) and H4 (H3) (B) showed a shift from high molecular weight (arrow) toward the low molecular weight region. However, this shift is not fully characteristic of the more extensive degree of nuclease digestion of new DNA, for the label did not accumulate in the monomer as shown in Fig. 3B. Instead, the dimer region contained a disproportionate amount of radioactivity; there is apparently a substantial portion of new histone associated with chromatin which is not assembled into typical nucleosomes, the actual structure of which awaits further examina-

A. T. Annunziato, R. K. Schindler, M. G. Riggs, and R. L. Seale, unpublished results.

A

D -

M[

B o b c d e f g h

D -

M L

FIG. 5. Newly synthesized proteins do not exchange during chromatin fractionation and electrophoresis. The S.1 fraction (see "Materials and Methods") from chromatin labeled for 2 min with ["Hlarginine and [:'Hllysine was mixed with the S.3, T.2, and T fractions from unlabeled chromatin. The mixed samples were subjected to electrophoresis and prepared for fluorography. Lane designations: a, labeled S.l control; b, unlabeled S.3; c, S.l/S.3, mixed in the presence of CB buffer plus 0.3 M NaCl and dialyzed 12 h against 2 mM EDTA; d, unlabeled T.2; e, S.l/T.2, combined; f, unlabeled T; g, S.I/T, combined; h, S.I/S.3 combined. The positions of mono- ( M ) and dinucleosomes (D) are indicated. A , ethidium bromide stain; B. fluorograph.

Dl i M O N O I

11

FIG. 6. Two-dimensional analysis of chromatin-associated newly synthesized proteins. Cells were labeled with ['Hlarginine for 2 min; nuclease-digested chromatin (prepared by nuclear lysis in 0.2 mM EDTA) was separated in a DNP gel in the first dimension, and in a SDS-polyacrylamide gel in the second dimension. A fluorograph is shown. The positions of mono- and dinucleosomes are indicated.

tion. From Fig. 7, we conclude that the shift in molecular weight of chromatin complexed with newly synthesized core histones is detectable, but neither of the magnitude, nor of the periodicity (45) expected for exclusive assembly onto newly synthesized DNA.

The behavior of newly synthesized H1 protein is opposite that of the new core histones (Fig. 7C). In fact, H1 is associated


with chromatin of a higher average molecular weight than total chromatin, and very little new H1 is associated with monomeric DNA.

In the second approach to this analysis, the SI1 and P chromatin fractions (46; see “Materials and Methods”) were analyzed by electrophoresis in DNP gels following stripping of nucleosomes of non-histone proteins and H1 in 0.45 M NaC1. Salt washing also removes nonassembled, newly synthesized histones which are predominantly H3, H4, and H2B (43), so the analysis of stripped chromatin is weighted for new H2A, 70% of new H2B, and less than half of new H3 and H4 (43). Thus, only stable, or fully assembled nucleosomal histones are measured, as compared to the analysis of Fig. 7 where the sum of both components was analyzed. The separation between bulk and newly synthesized DNA of stripped nucleosomes from the SI1 fraction is shown in Fig. 8A. The newly replicated DNA was degraded more rapidly, resulting in its depletion from the high molecular weight region of the gel (arrow), and its corresponding enrichment in the low molecular weight region. A second feature diagnostic for new chromatin DNA is the shortened subunit repeat, evident in the phasing difference between nucleosomal multimers of new and old chromatin DNP and DNA (11, 28, 32, 45, 50, 55).

These criteria were then applied to follow the fate of newly synthesized proteins by pulse-labeling [‘*C]thymidine-prelabeled cells for 2 min with [3H]lysine. Electrophoretic separation of the stripped nucleosomes ( i e . stable new core histones) showed that the core histones were slightly out of phase in the dimer and trimer regions (shaded areas of Fig. 8B), but this difference consisted of a shoulder, not as a completely shortened repeat, expected for deposition on new DNA (Fig. 8A). The second important feature of this profie was that, as previously shown in Figs. 4 and 7 for unstripped chromatin, there was no diminution of pulse-labeled protein radioactivity from the high molecular weight region (arrow, Fig. 8B) as seen for new chromatin DNA (arrow, Fig. a). Thus, the new histones which were as salt-resistant as mature nucleosomal histones showed characteristics of association with both nonreplicating and new DNA. The low yield of monomer (Fig. 8B) reflects a shortcoming of the methodology used to sepa-

d m

FIG. 7. Distribution of individual newly synthesized histones relative to individual total histone in DNP gels. Proteins were labeled and separated for two-dimensional analysis as in Fig. 6. The histone bands were scanned along their long axes. Solid line,

the first dimension DNP gel is from left to right. A , histone H2A B , Coomassie blue stain; dotted line, fluorographic image. Migration in

histone H3; C, H1. Arrow indicates position of depletion of new chromatin in the multimer region due to its nuclease sensitivity. Profile of H2A was similar to that of H2B, and that of H3 was similar to H4. The positions of mono- (m) and dinucleosomes ( d ) are indicated.

FRACTION

FIG. 8. Distribution of pulse-labeled histones in stripped chromatin. [’4C]Thymidine-prelabeled cells were incubated with [3H]thymidine (A) or r3H]lysine (B) for 2.5 min; the SI1 chromatin fraction was stripped of non-histones and H1 in 0.45 M NaCl (see “Materials and Methods”), and separated in DNP gels; gels were sliced into 1-mm sections. 0, I4C; 0, 3H. Migration is from Left to right. The positions of the mono- (m) and dinucleosomes ( d ) are indicated. Arrow indicates the area of depletion of newly replicated chromatin DNA.

rate chromatin from salt-dissociated proteins, for monomeric nucleosomes do not pelletize well; however, the yield of monomer does not affect this analysis.

Within 15 min the migration of new core histones in DNP gels was identical to that of bulk chromatin (data not presented). Also, analysis of 0.45 M NaCl solubilized chromatin from the P fraction yielded identical results. We interpret the new core histone profile of Fig. 8B to represent the sum of two overlapping curves representing association of new core histones with both nonreplicating and newly replicated chromatin DNA. These data are in full agreement with experiments employing salt elution as the fist stage of separation of new from bulk chromatin (this study), and with other electrophoretic analyses of histone deposition (55).

DISCUSSION

In this communication, we have attempted to determine the site of assembly of newly synthesized histones not only to resolve the conflicting reports in the literature surrounding this topic, but also because the solution to this question bears heavily on the understanding of other chromatin functions, including the determination, maintenance, and inheritance of both positive and negative transcriptional control elements. To state the importance of this problem succinctly, if it is shown that some core histones are assembled at sites other than those on newly replicated DNA, then it follows that core histones (perhaps histone octamers) redistribute, instead of occupying fixed positions. Depending on the degree of core histone redistribution, it may be necessary to revise theories of cell differentiation and transcriptional control mechanisms based on stable positioning and inheritance of these epigenetic agents.

The dual nature of the fractionation behavior of the core histones and the proportions thereof strongly indicate their existence in two fundamentally different structures. If new histones were exclusively and stoichiometrically assembled on new DNA, then the newly synthesized core histones should


follow only the properties of newly replicated chromatin DNA (see Introduction). We do not observe this.

Since parental histone octamers are distributed conserva- tively at the replication fork (7, 30-32), only one daughter DNA duplex is available for de nouo nucleosome assembly (7). Because both daughter DNA duplexes are equally labeled with thymidine, it is clear that at most 50% of pulse-labeled DNA can become associated with newly assembled nucleosomes. Since both arms of the replication fork may not have identical properties, we have followed the fractionation of both new DNA and new histones in all fractions in chromatin isolation procedures, including the final nuclear residue.

In an earlier report (44), we found that nascent chromatin DNA was several-fold enriched over bulk DNA in the fractions eluted at 0.4-0.6 M NaC1, and together with the insoluble pellet, contained 30-50% of the nascent DNA, varying with the experiment. We speculated that these nascent chromatin fractions might be derived from the unassembled DNA duplex; the present data that newly synthesized core histones are equally enriched in these fractions support this interpre- tation. However, if these new histones are associated with nascent DNA, the structures are not typical nucleosomes (44) and, therefore, must be considered as intermediates in the assembly process (43). It is now apparent that newly deposited histones and mature nucleosomal histones possess very different properties (43) reflecting stages in somatic cell nucleosome assembly (6, 33, 43), as contrasted to the apparent single-step assembly of embryonic systems (51).

Because salt concentrations sufficiently high to release the assembly intermediate chromatin also promote dissociation of a substantial fraction of newly synthesized core histones (43), we have been unable to unambiguously demonstrate that the correlation of new histones with new DNA in these fractions represents their assembly together. From the yields, however, we are able to state that the upper limit of this possible association is approximately 50% of new core histones with new DNA. This correlation, while not exclusive, is highly preferential, for only 0.5% of the DNA by mass is replicated during the labeling interval.

New histones eluted from nuclei under conditions of low to moderate ionic strength do not co-electrophorese with the nascent chromatin DNA in those fractions. Because core histone exchange during experimental manipulation at the lower ionic strengths was excluded, the deposition of new histones onto nonreplicating chromatin is indicated. Compa- rable conclusions have been reported in other studies (11, 13, 14,34,45,55). We conclude that while a significant proportion of new core histone may be associated with new DNA, this association accounts for approximately half of the new protein, and is not exclusive. New histones are also deposited onto nonreplicating chromatin,

In contrast to nascent core histones, newly synthesized H1 appears to be deposited solely onto bulk chromatin. Moreover, H1 was not deposited onto bulk chromatin en route to preferential assembly onto new DNA; after 15 min of labeling the elution profde of the newly synthesized H1 histones remained unaltered (data not presented). It has been reported that salt- induced H1 rearrangement can occur at salt concentrations of 70-140 II~M (36). In experiments performed in the absence of salt (this study), and in which protein-DNA complexes were chemically cross-linked prior to chromatin isolation (13, 14), the selective association of new H1 with nonreplicating chromatin was still observed.

In a set of companion experiments which also served as controls for chromatin prepared without exposure to salt, 1) the fractionation of new DNA and new histones after nuclease digestion, 2) the enhanced nuclease sensitivity, and 3) the

shorter repeat length inherent to nascent chromatin DNA (28-30, 32, 45, 50, 55) were used as criteria to determine the site of new core histone assembly. These experiments corrob- orated those employing the salt elution procedure; evidence was found consistent with bimodal distribution of new histones to both newly replicated and nonreplicating DNA (13, 14,55). A greater abundance of H3 and H4 histones over H2A and H2B correlating with new DNA was found, but was less pronounced than in studies performed with metabolic inhibitors (13). H2A and H2B entered mature chromatin directly without passing through assembly intermediate stages (6, 13, 14, 43). Histone H1 showed no detectable correlation with new DNA 13, 14, 55).

A large measure of earlier discord on the site of histone deposition can be ascribed to the observation that assembly occurs on both types of DNA. Thus, procedures that enrich for one species uersus the other would yield opposite results. In one study, only IO-50% of chromatin was solubilized and analyzed, while the rest was discarded (6); in other reports also, only soluble chromatin was analyzed, and the yields of radioactivity were not given (5,8-11,33). In the present study, we have accounted for and identified the radioactivity in new DNA and in the individual new histones in all fractions obtained through chromatin manipulations. By doing so, we have confmed the evidence for both assembly sites, and we believe this to be the source of disagreement on this topic.

Labeling and fractionation procedures in early reports of new histone assembly on nonreplicating chromatin (8, 9) probably overestimated the proportion of protein accumulat- ing on nonreplicating DNA due to the labeling procedure. Approximately 30% of labeled lysine enters histone H1 which is now known to be deposited independently from core histones. However, a later analysis utilizing stripped nucleosomes (10) did c o n f m these early experiments.

Upon close examination, the literature is in rather close agreement on the topic of bimodal histone deposition. Chestier and Yaniv (42) estimated that 30-50% of new histone is deposited on new DNA in SV40. While quantitative data were not presented, similar relative proportions can be deduced from the data of Coca-Prados et aZ. (35) in SV40, and in MH- 134SC cells by Senshu et aZ. (12). Other recent studies in cellular systems are in accord that new histones are deposited on both newly replicated and on nonreplicating DNA (IO, 13, 14, 34, 45, 55).

Several laboratories have reported the differential deposition of H3 and H4 uersus H2A and H2B (6, 13, 14, 43, 55). This presents an interpretative enigma for core particle assembly. The data on core particle assembly utilizing dense amino acid incorporation were taken to indicate exclusive assembly from new histones (47). However, considerable deviation from exclusive assembly could have occurred without detection in that study, since the labeling was performed with ["Hllysine, and total chromatin, including non-histone proteins and HI, was banded. If new histones were assembled exclusively into octamers, then there should not be disparate sites of deposition of histone pairs. Worcel et al. (6) interpreted this phe- nomenon to indicate sequential assembly; H3 and H4 are deposited initially, later followed by the association of new H2A and H2B into the same structures. If such were the case, then a long labeling period should show stoichiometry among newly assembled core histones, but evidence does not support this prediction. Senshu et al. (12) found an enrichment for H3 and H4 over H2A and H2B in chromatin replicated in 1-h labeling protocols. In SV40, H3 and H4 are preferentially deposited on replicative intermediates, while H2B is deposited in physically distinct minichromosomes (33). Further, pulse- labeled histones are not stoichiometric either in the assembled


or in the assembly intermediate state (43). Recent evidence 83-88 indicates that hybrid nucleosomes can be constructed from 21. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56,

both new and Old histones (52)' and that histones can enter 22. Kuehl, L., Lyness, T., Dixon, G . H., and Levy-Wilson, B. (1980) J. chromatin in the absence of DNA replication (37-41). These data have imp1ications for 23. Ohlenbusch, H. H., Olivera, B. M., Tuan, D., and Davidson, N.

function. During the cell cycle, the doubling of DNA mass is (1967) J. Mol. Biol. 25. 299-315

335-341

Biol. Chem. 255,1090-1095

matched by that of the histones. If new histones were exclusively assembled on new DNA, then their positions on DNA might be viewed as stable. Since deposition is not exclusive, then perhaps half of the sites on new DNA must be fiied with pre-existing histones. The movement of histones on DNA, thus, is a subject on which we know little, and is crucial to understanding genomic function. For instance, the active state in chromatin may reflect the ability of nucleosomes to be dislocated by polymerases, rather than to be transcribed per se. The theory that the lineage of cell determination is maintained by transmission from parent to daughter chromatids of specific nucleosome types permanently affixed to their binding sites on DNA (49) would have to be modified to allow for histone redistribution, and for continual remodeling of gene expression states. Recent studies by Jackson and Chalkley (14) and by Fowler et al. (56) present rather strong evidence for the redistribution of core histones from their original site of deposition on DNA.

REFERENCES 1. Edenberg, H. J., and Huberman, J . A. (1975) Annu. Reu. Genet.

2. Seale, R. L. (1978) in The Cell Nucleus (Busch, H., ed) Vol. 4, pp.

3. DePamphilis, M. L., and Wassarman, P. M. (1980) Annu. Reu.

4. Tsanev, R., and Russev, G. (1974) Eur. J. Biochem. 43,257-263 5. Cremisi, C., Chestier, A., and Yaniv, M. (1977) Cell 12, 947-951 6 . Worcel, A., Han, S., and Wong, M. L. (1978) Cell 15, 969-977 7. Seidman, M. M., Levine, A. J., and Weintraub, H. (1979) Cell 18,

8. Seale, R. L. (1976) Proc. Natl. Acad. Sci. U. S. A. 73,2270-2274 9. Jackson, V., Granner, D., and Chalkley, R. (1976) Proc. Natl.

10. Hancock, R. (1978) Proc. Natl. Acad. Sci. U. S. A. 75, 2130-2134 11. Murphy, R. F., Wallace, R. B., and Bonner, J. (1980) Proc. Natl.

12. Senshu, T., Fukuda, M., and Ohashi, M. (1978) J . Biochem.

13. Jackson, V., and Chalkley, R. (1981) Cell 23, 121-134 14. Jackson, V., and Chalkley, R. (1981) J. Biol. Chem. 256,

15. Berezney, R., and Coffey, D. S. (1975) Science (Wash. DC) 189,

16. Dijkwel, P. A., Mullenders, L. H. F., and Wanka, F. (1979) Nucleic

17. Pardoll, D. M., Vogelstein, B., and Coffey, D. S. (1980) Cell 19,

18. Sanders, M. M. (1978) J. Cell Biol. 79, 97-109 19. Thomas, J. O., and Kornberg, R. D. (1975) Proc. Natl. Acad. Sci.

20. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,

9,245-284

155-172, Academic Press, New York

Biochem. 49,627-666

439-449

Acad. Sci. U. S. A. 73,2266-2269

Acad. Sci. U. S. A. 77,3336-3340

(Tokyo) 84,985-988

5095-5103

291-293

Acids Res. 6, 219-230

527-536

U. S. A. 72,2626-2630

24. More, I: A. R., and Paul, J. (1973) Exp. Cell Res. 76, 79-86 25. Davie, J. R., and Candido, E. P. M. (1980) FEBS Lett. 110,

26. Seale, R. L., and Simpson, R. T. (1975) J. Mol. Biol. 94,479-501 27. Hildebrand, C. E., and Walters, R. A. (1976) Biochem. Biophys.

28. Levy, A., and Jakob, K. M. (1978) Cell 14,259-267 29. Klempnauer, K.-H., Fanning, E., Otto, B., and Knippers, R. ( 1980)

J. Mol. Biol. 136,359-374 30. Seale, R. L. (1976) Cell 9,423-429 31. Weintraub, H. (1976) Cell 9,419-422 32. Seale, R. L. (1978) Proc. Natl. Acad. Sci. U. S. A. 75,2717-2721 33. Cremisi, C., and Yaniv, M. (1980) Biochem. Biophys. Res. Com-

34. Annunziato, A. T., and Woodcock, C. L. F. (1981) Prog. Nucleic

35. Coca-Prados, M., Vidali, G., and Hsu, M. T. (1980) J . Virol. 36,

36. Caron, F., and Thomas, J. 0. (1981) J . Mol. Bwl. 146,513-537 37. Nadeau, P., Oliver, D. R., and Chalkley, R. (1978) Biochemistry

38. Russev, G., Vassiliv, L., and Tsanev, R. (1980) Nature 285,

39. Sheinin, R., Setterfield, G., Dardick, I., Kiss, G., and Dubsky, M.

40. Zlatanova, J. (1980) Mol. Cell. Biochem. 35,49-53 41. Groppi, V. E., Jr., and Coffino, P. (1980) Cell 21, 195-204. 42. Chestier, A., and Yaniv, M. (1979) Proc. Natl. Acad. Sci. U. S. A.

43. Seale, R. L. (1981) Biochemistry 20, 6432-6437 44. Annunziato, A. T., Schindler, R. K., Thomas, C. A,, Jr., and Seale,

R. L. (1981) J. Biol. Chem. 256, 11880-11886 45. Seale, R. L. (1981) Prog. Nucleic Acid Res. Mol. Biol. 26, 123-134 46. Todd, R. D., and Garrard, W. T. (1977) J. Bwl. Chem. 252,

47. Leffak, I. M., Grainger, R., and Weintraub, H. (1977) Cell 12, 837-845

48. Albright, S. C., Wiseman, J. M., Lange, R. A,, and Garrard, W. T. (1980) J. Bwl. Chem. 255,3673-3684

49. Weintraub, H., Flint, S. J., Leffak, I. M., Groudine, M., and Grainger, R. M. (1978) Cold Spring Harbor Symp. Quant. Biol.

50. Murphy, R. F., Wallace, R. B., and Bonner, J. (1978) Proc. Natl.

51. Laskey, R. A., and Earnshaw, W. C. (1980) Nature 286, 763-767 52. Russev, G., and Hancock, R. (1981) Nucleic Acids Res. 9,

53. Herman, T. M., DePamphilis, M. L., and Wassarman, P. M.

54. Cusick, M. E., Herman, T. M., DePamphilis, M. L., and Wassar-

55. Jackson, V., Marshall, S., and Chalkley, R. (1981) Nucleic Acids

56. Fowler, E., Farb, R., and El-Saidy, S. (1982) Nucleic Acids Res.

164-168

Res. Commun. 73, 157-163

m ~ n . 92, 1117-1123

Acid Res. Mol. Biol. 26, 135-149

353-360

17,4885-4893

584-586

(1980) Can. J. Biochem. 58, 1359-1369

76,46-50

4729-4738

42,401-407

Acad. Sci. U. S. A. 75,5903-5907

4129-4137

(1979) Biochemistry 18,4563-4571

man, P. M. (1981) Biochemistry 20,6648-6658

Res. 9, 4563-4581

10, 735-748

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