Cell, Vol.
12,947-951,
December
1977, Copyright
0 1977 by MIT
Preferential Association of Newly Synthesized Histones with Replicating SV40 DNA Chantal Cremisi, Annick Moshe Yaniv Department of Molecular lnstitut Pasteur 75015 Paris, France
Chestier
and
Biology
Summary The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA. Introduction Replication in eucaryotic cells is a complex process that involves not only duplication of the DNA, but also the coordinate synthesis of histones with the subsequent formation of the nucleosome. The steps of DNA replication and histone synthesis have been studied separately, but little is known about their intimate association during replication. We chose to study the replication of SV40 chromosomes as a simple model system for eucaryotic chromosome replication. Viral DNA synthesis, similarly to DNA synthesis in eucaryote cells, is a discontinuous process: 4s DNA fragments (150200 nucleotides long) are synthesized in ~15 set on both strands at the level of the replication fork (Salzman et al., 1973). Like cellular DNA, viral DNA is associated with histones in the nuclei; these nucleoprotein complexes (minichromosomes) of SV40, polyoma or papilloma viruses exhibit a chromatin-like structure in the form of a beaded ring (Griffith, 1975; Cremisi et al., 1976; Favre et al., 1977). The viral DNA remains associated with proteins, possibly histones during replication, since the density of the replicative intermediates and mature nucleoprotein complexes is identical (Hall, Meinke and Goldstein, 1973; Cremisi et al., 1976). The mode of formation of nucleosomes during viral DNA replication, however, is still unsolved. Are histones effectively present on the replicative DNA intermediates? Do the newly synthesized histones associate preferentially with the newly replicated DNA or is there a random distribution of these histones between replicating and nonreplieating viral chromosomes? Conflicting results have
been obtained concerning the replication of the cellular chromatin. Jackson, Granner and Chalkley (1975, 1976) and Seale (1976) suggested that newly synthesized histones do not associate preferentially with the newly replicated DNA, whereas Weintrau b (1973)) Tsanev and Russev (1974)) and Freedlender, Taichman and Smithies (1977) suggest the opposite. Studies have been carried out on the SV40 replication system in an attempt to solve some of these problems. Evidence is provided suggesting that newly synthesized histones associate preferentially to the newly replicated DNA. Results
and Discussion
Biochemical studies of the SV40 nucleoprotein complexes extracted from infected cells have shown that they contain the four cellular histones H2a, H2b, H3 and H4; histone HI is found depending upon the ionic strength of extraction (Varshavsky et al., 1976). SV40 DNA requires about lo-20 min to replicate in infected nuclei (Levine, Kang and Billheimer, 1970). By short pulse labeling and zonal sedimentation, it is possible to label the replicating form preferentially and to separate it from mature complexes (Hall et al., 1973; Cremisi et al., 1976; Figure 1). If a preferential association occurs between newly synthesized histones and replicating DNA, we predict that the majority of histones labeled for 10 min would be associated with the replicative DNA. If, however, the opposite is true- random association of newly synthesized histones-we expect to find those histones labeled for 10 min associated with all viral DNA (mature and replicating). As described in Experimental Procedures, infected cells were labeled for either 10 min or 16 hr with 3H-thymidine and 35S-methionine. Nuclei were isolated in the presence of 0.5% NP40, and the nucleoprotein complexes were extracted with 0.25% Triton X-100 in the absence of bivalent cations. The majority of the viral DNA labeled for 16 hr sediments as a symmetric peak as shown in Figure 1. Electron microscope screening of both gradients represented in Figure 1 (short and long labeled cultures) showed that the concentration of the viral minichromosomes parallels the 16 hr 3H-thymidine labeling pattern (results not shown). The gradients were subdivided into six sections, as shown in Figure 1, in order that fraction II contain the replicating DNA, and fraction IV the mature DNA. The material was concentrated by high speed centrifugation, and the histone content was analyzed by polyacrylamide gel electrophoresis either in the presence of SDS or in the presence of acetic acid and urea. Staining of the gels (Figures 2A and 2C) shows that the majority
Cell 948
of the histones are present in section IV, in accordance with the sedimentation and the electron microscope observations. On the other hand, the
autoradiograms (Figures 2B and D) show that the majority of the pulse-labeled ?S histones are in section II, the fraction rich in replicative DNA. When complexes were labeled for 16 hr with 35Smethionine, the staining and the autoradiography patterns of histones were identical with the maximal concentration in section IV. The staining patterns of both the SDS and the acidic urea gels when compared with histone markers prove unambiguously that the bands observed are indeed histones Hl, H2b, H2a, H3 and H4. The autoradiography reveals histones H3, H2b and H4 only, since Hl and H2a lack methionine. We observe that histone Hl is associated with the viral minichromosomes (Figure 2C); we cannot, however, prove that this association is physiological. No attempt was made in these studies to follow the distribution of newly synthesized histone Hl. Table 1 summarizes the ratio between the radioactive and nonradioactive histone concentrations as recorded from the autoradiograms and from the stained gels. It clearly demonstrates that the specific activity of the histones decreases at least 8 fold between replicative and mature complexes. From these results, we conclude that newly synthesized histones (except Hl) are preferentially associated with the replicative intermediates and do not exchange with the majority of the histones present on the mature complexes. We presume that histone H2a follows the same pattern as the other three histone components of the nucleosome core. Our electron microscope observations have shown that SV40 replicative intermediates indeed contain nucleosomes on prereplicative and on both daughter double helices (unpublished observations). These findings are in agreement with the observations of McKnight and Miller (1977) on Drosophila melanogaster embryo chromatin. To determine whether newly synthesized histones preferentially exchange with the bulk of the histones of the cellular chromatin that comprises the majority of unlabeled histones in the infected cell, we solubilized this fraction and measured the concentration of 35S histones and 3H-DNA. Since the viral infection stimulates cellular DNA synthesis, it is obvious that labeled histones are found
1
FRACTION NUMBER -----1
II
Figure 1. Sedimentation cleoprotein Complexes
UIEP
Analysis
PI
of Mature
and
Replicative
Nu-
(a) CVI cells were infected with a multiplicity of 50 PFU per cell. 41 hr post-infection, the cultures were incubated for 20 min with fresh medium lacking methionine, followed by labeling for 10 min with 1 mCi 55S-methionine (580 Cilmmole) and 0.5 mCi (45 Ci/mmole) of H3-thymidine in 0.5 ml of methionine-free medium. The cells were then washed twice with cold PBS, and the SV40 minichromosomes were extracted and purified by centrifugation in an SW41 rotor at 40,000 rpm for 110 min. Fractions were collected, and aliquots were precipitated and counted. The complexes from different positions of the gradients I-VI as indicated in the figure were pooled, and the complex was pelleted by centrifugation for 14 hr at 50,000 rpm (O0). (b) CVI cells were infected as above and labeled between 32-41 hr post-infection with 100 &i of 35S-methionine and 100 &i of 3H-thymidine in a medium containing l/50 of the normal methionine concentration (x-x).
Figure
2. Analysis
of Histones
Bound
to Mature
and Replicating
DNA
(A and B) SDS-polyacrylamide gel. The complex fractions concentrated by sedimentation (Figure 1) were dissolved in sample buffer and electrophoresed in 17.5% polyacrylamide discontinuous gels according to Blatter et al. (1972). Gels were stained with Coomassie blue, photographed, dipped in DMSO containing PPO and autoradiographed as described by Bonner and Laskey (1974) and Laskey and Mills (1975). (A) stained gel; (B) autoradiogram. (a) histone Hi, sections I-VI from Figure 1; (b) total calf thymus histones; (c) calf thymus histones H3, H2b, H2a and H4; (d) polyoma virus labeled with C’* amino acids. (C and D) Acidic urea polyacrylamide gels. The complex fractions were treated with a buffer containing IO M urea, 0.9 M acetic acid, 5 mg/ml protamine and 1% (w/v) p-mercaptoethanol for 18 hr at 20°C (Schaffhausen and Benjamin, 1976). The samples were separated on 14 cm, 15% gels containing 0.9 M acetic acid and 2.5 M urea as described by Panyim and Chalkley (1969). (C) stained gel; (D) autoradiogram. (a) CVl chromatin, sections I-VI from Figure 1; (b) CVl chromatin and calf thymus histones; (c) histone Hl; (d) calf thymus histones H3, H2b. H2a; (e) calf thymus histones.
Histone 949
a
Association
I II
I
II
with
Replicating
DNA
III
IV
v
VI
Ill
IV
v
VI
b c d e
a
i ii
iii
IV
v
vl’b
c d
Cell 950
Table 1. Histone Gradient
Concentration
in the Different
Sections
of the
Section
I
II
III
IV
Autoradiography (Arbitrary Units)
30
75
69
44
Units)
5
17
53
61
Autoradiography Staining
6
Staining (Arbitrary Ratio
4.4
1 .3
SDS gels (staining and autoradiography) were scanned, the intensitv of bands is expressed in arbitrarv units.
0.72 and
associated with the cellular chromatin. If newly synthesized histones incorporate preferentially into the cellular chromatin, however, we expect that the major fraction of %-methionine-labeled histones are associated with the cellular chromatin, and that the ratio of 35S/3H in the chromatin pellet greatly exceeds that of the viral replicating chromosomes. When measured, this ratio was roughly equal to that of replicating viral chromosomes, excluding the preferential exchange with nonreplieating cellular chromatin. The experiments described in this work analyze only the newly synthesized histones and are confined to a very short time lapse in the viral growth cycle; the cells were pulse-labeled for periods shorter than one viral DNA replication round. We cannot exclude a slow exchange of histones between cellular and viral chromatins occurring throughout the viral growth cycle. In fact, studies on histones associated with polyoma virus showed that a small fraction of cellular histones labeled before the infection are found associated with mature virions (Frearson and Crawford, 1972; Fey and Hirt, 1974). These experiments analyze the entire pool of the viral DNA synthesized over a time span of 2-6 days. At least two hypotheses can be advanced to explain these findings. First, the incorporation of prelabeled cellular histones into viral DNA may involve some exchange of viral and cellular parental histone cores due to a close “functional” association between cellular and viral replicons in the nuclei (LeBlanc and Singer, 1974). Second, during transcription and replication of cellular and viral chromatin, some nucleosomes dissociate and permit a slow histone exchange over a long time span compared to the viral DNA replication cycle. Our results on the preferential association of newly synthesized histones and DNA are in contradiction with the findings of Jackson et al. (1975, 1976) and Seale (1976). who found by density labeling that there was a random distribution of newly synthesized histones. These investigators, however, made several assumptions in the interpretation of their results that were not fully proven.
The possibility that the mechanism of cellular and viral chromatin replication differs extensively seems to us improbable, since the virus utilizes mainly the cellular replication system and coordinately stimulates cellular DNA and histone synthesis. Experlmental
Procedures
Cells and Virus Intaction African green monkey kidney cells (line CVI) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum in 88 mm plastic dishes. Plaque-purified SV40 was used to infect the cell cultures at an input multiplicity of 3050 PFU per cell. At the end of a 2 hr adsorption period, 10 ml of Dulbecco’s modified Eagle’s medium containing 1% serum were added to each dish. Purifkatbn of Nucleoprotein Complexes At 41 hr post-infection, the monolayer was washed twice with phosphate buffer (PBS), the nuclei were isolated in the presence of 0.5% NP40 and the nucleoprotein complex was extracted with 0.2% Triton and 0.2 M NaCl (Green, Miller and Hendler, 1971). Two successive extractions were performed for 30 min at 20°C. The Triton supernatant was obtained after centrifugation at 800 x g, and the nucleoprotein complexes were partially purified by zonal sedimentation on 5-20% (w:w) sucrose gradients in 0.01 M Tris-HCI (pH 7.9), 0.061 M EDTA, 0.2 M NaCl. Gel Electrophoresls Samples from sucrose gradients were concentrated by high speed sedimentation and prepared for analysis of histones. Two different 14 cm long slab gels were used: 17.5% polyacrylamide (Blatter et al., 1972) and acidic urea 15% polyacrylamide gels (Panyim and Chalkley. 1969). In this case, complex fractions were treated with a buffer containing 10 M urea, 0.9 M acetic acid, 5 mg/ml protamine sulfate and 1% (w/v) &mercaptoethanol for 16 hr at 20°C (Schaffhausen and Benjamin, 1978). SDS gels were stained for 1 hr with Coomassie blue and the acidic urea gel with amido black for at least 5 hr. For autoradiography. the gels were impregnated with PPO (Bonner and Laskey. 1974; Laskey and Mills, 1975), dried and clamped to hypersensitized film for exposure at -70°C. Acknowledgments This work was supported by grants from the CNRS, INSERM and the Fondation pour la Recherche Medicale Francaise. We are indebted to Ddile Croissant for providing us with electron microscope facilities, C. Maczuka for help in preparing the manuscript and G. Butler-Browne for valuable criticisms of the manuscript. 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 18 U.S.C. Section 1734 solely to indicate this fact. Received
July 18, 1977;
revised
August
22, 1977
References Blatter, D. P.. Garner, F.. Van Slyke, Chromatography 64, 147-151. Bonner, 88.
M. W. and Laskey.
R. A. (1974).
Cremisi. C., Pignatti, Virol. 77, 204-211.
P. F., Croissant,
Favre, M.. Breitburd, Virol. 27, 1205-1209.
F., Croissant,
Fey, G. and Hirt, Biol. 39, 235-341.
B. (1974).
K. and Bradley,
Cold
A. (1972).
Eur. J. Biochem. 0. and Yaniv.
0. Spring
and
Orth,
Harbor
J.
46,83-
M. (1978).
J.
G. (1977).
J.
Symp.
Quant.
Histone 951
Association
Frearson, 141-155.
with
P. M. and
Replicating
Crawford,
L. V. (1972).
Freedlender, F. E., Taichman, chemistry 16, 1802-1808.
L. and
Green, M. H., Miller, H. I. and Acad. Sci. USA68, 1032-1038. Griffith,
J. D. (1975).
Science
Hall, M. R., Meinke, 901-908.
DNA
J. Gen.
Smithies,
Hendler,
Viral.
74.
0.
(1977).
Bio-
S. (1971).
Proc.
Nat.
187, 1202-1203. (1973).
J. Virol.
Jackson, V., Granner, D. K. and Chalkley, Acad. Sci. USA 72, 4440-4444.
R. (1975).
Proc.
Nat.
Jackson, V.. Granner. D. K. and Chalkley, Acad. Sci. USA 73, 2288-2269.
R. (1978).
Proc.
Nat.
Laskey, 344.
R. A. and
W. and Goldstein,
Mills,
LeBlanc. D. J. and USA 71, 2236-2240.
Singer,
Levine, A. J., Kang. Biol. 50, 549-568. f&Knight,
A. D. (1975).
Chalkley,
Eur. J. Biochem.
M. F. (1974).
M. S. and
S. L. and Miller,
Panyim, S. and 130, 337-346.
D.A.
Proc.
Billheimer,
56, 335Acad.
F. E. (1970).
0. L.. Jr. (1977). R. (1969).
Nat.
Seale,
R. L. (1978).
Tsanev.
Proc.
R. and Russev.
Nat. Acad. G. (1974).
Cell 72, 795804.
Arch.
Biochem.
T. L. (1976).
Biophys.
H. (1973).
Cold
Spring
Proc.
M. M.
Nat. Acad.
Sci. USA 73, 2270-2274. Eur. J. Biochem.
Varshavsky, A. J.. Bakayer. V. V., Chumackov, giev.G. P. (1976). Nucl. Acids Res.3, 2101-2113. Weintraub. 247-256.
Sci.
J. Mol.
Salzman. N. P.. Fareed, G. C.. Sehing, E. D. and Thoren. (1973). Cold Spring Harbor Symp. Quant. Biol. 38, 257-285. Schaffhausen. B. S. and Benjamin, Sci. USA 73, 1092-1098.
12,
Harbor
Symp.
43, 257-283. P. M. and Geor/ Quant.
Biol. 38,
12,947-951,
December
1977, Copyright
0 1977 by MIT
Preferential Association of Newly Synthesized Histones with Replicating SV40 DNA Chantal Cremisi, Annick Moshe Yaniv Department of Molecular lnstitut Pasteur 75015 Paris, France
Chestier
and
Biology
Summary The assembly of newly synthesized histones into nucleosomes during replication of SV40 minichromosomes in vivo was studied. Infected cells were labeled with 35S-methionine for a time shorter than that required to complete a round of viral DNA replication. Mature and replicating SV40 minichromosomes were extracted and separated by zonal sedimentation, and their histone content was analyzed by polyacrylamide gel electrophoresis (SDS and acidic urea). We show that the pulse-labeled histones associate preferentially with the replicating DNA. Introduction Replication in eucaryotic cells is a complex process that involves not only duplication of the DNA, but also the coordinate synthesis of histones with the subsequent formation of the nucleosome. The steps of DNA replication and histone synthesis have been studied separately, but little is known about their intimate association during replication. We chose to study the replication of SV40 chromosomes as a simple model system for eucaryotic chromosome replication. Viral DNA synthesis, similarly to DNA synthesis in eucaryote cells, is a discontinuous process: 4s DNA fragments (150200 nucleotides long) are synthesized in ~15 set on both strands at the level of the replication fork (Salzman et al., 1973). Like cellular DNA, viral DNA is associated with histones in the nuclei; these nucleoprotein complexes (minichromosomes) of SV40, polyoma or papilloma viruses exhibit a chromatin-like structure in the form of a beaded ring (Griffith, 1975; Cremisi et al., 1976; Favre et al., 1977). The viral DNA remains associated with proteins, possibly histones during replication, since the density of the replicative intermediates and mature nucleoprotein complexes is identical (Hall, Meinke and Goldstein, 1973; Cremisi et al., 1976). The mode of formation of nucleosomes during viral DNA replication, however, is still unsolved. Are histones effectively present on the replicative DNA intermediates? Do the newly synthesized histones associate preferentially with the newly replicated DNA or is there a random distribution of these histones between replicating and nonreplieating viral chromosomes? Conflicting results have
been obtained concerning the replication of the cellular chromatin. Jackson, Granner and Chalkley (1975, 1976) and Seale (1976) suggested that newly synthesized histones do not associate preferentially with the newly replicated DNA, whereas Weintrau b (1973)) Tsanev and Russev (1974)) and Freedlender, Taichman and Smithies (1977) suggest the opposite. Studies have been carried out on the SV40 replication system in an attempt to solve some of these problems. Evidence is provided suggesting that newly synthesized histones associate preferentially to the newly replicated DNA. Results
and Discussion
Biochemical studies of the SV40 nucleoprotein complexes extracted from infected cells have shown that they contain the four cellular histones H2a, H2b, H3 and H4; histone HI is found depending upon the ionic strength of extraction (Varshavsky et al., 1976). SV40 DNA requires about lo-20 min to replicate in infected nuclei (Levine, Kang and Billheimer, 1970). By short pulse labeling and zonal sedimentation, it is possible to label the replicating form preferentially and to separate it from mature complexes (Hall et al., 1973; Cremisi et al., 1976; Figure 1). If a preferential association occurs between newly synthesized histones and replicating DNA, we predict that the majority of histones labeled for 10 min would be associated with the replicative DNA. If, however, the opposite is true- random association of newly synthesized histones-we expect to find those histones labeled for 10 min associated with all viral DNA (mature and replicating). As described in Experimental Procedures, infected cells were labeled for either 10 min or 16 hr with 3H-thymidine and 35S-methionine. Nuclei were isolated in the presence of 0.5% NP40, and the nucleoprotein complexes were extracted with 0.25% Triton X-100 in the absence of bivalent cations. The majority of the viral DNA labeled for 16 hr sediments as a symmetric peak as shown in Figure 1. Electron microscope screening of both gradients represented in Figure 1 (short and long labeled cultures) showed that the concentration of the viral minichromosomes parallels the 16 hr 3H-thymidine labeling pattern (results not shown). The gradients were subdivided into six sections, as shown in Figure 1, in order that fraction II contain the replicating DNA, and fraction IV the mature DNA. The material was concentrated by high speed centrifugation, and the histone content was analyzed by polyacrylamide gel electrophoresis either in the presence of SDS or in the presence of acetic acid and urea. Staining of the gels (Figures 2A and 2C) shows that the majority
Cell 948
of the histones are present in section IV, in accordance with the sedimentation and the electron microscope observations. On the other hand, the
autoradiograms (Figures 2B and D) show that the majority of the pulse-labeled ?S histones are in section II, the fraction rich in replicative DNA. When complexes were labeled for 16 hr with 35Smethionine, the staining and the autoradiography patterns of histones were identical with the maximal concentration in section IV. The staining patterns of both the SDS and the acidic urea gels when compared with histone markers prove unambiguously that the bands observed are indeed histones Hl, H2b, H2a, H3 and H4. The autoradiography reveals histones H3, H2b and H4 only, since Hl and H2a lack methionine. We observe that histone Hl is associated with the viral minichromosomes (Figure 2C); we cannot, however, prove that this association is physiological. No attempt was made in these studies to follow the distribution of newly synthesized histone Hl. Table 1 summarizes the ratio between the radioactive and nonradioactive histone concentrations as recorded from the autoradiograms and from the stained gels. It clearly demonstrates that the specific activity of the histones decreases at least 8 fold between replicative and mature complexes. From these results, we conclude that newly synthesized histones (except Hl) are preferentially associated with the replicative intermediates and do not exchange with the majority of the histones present on the mature complexes. We presume that histone H2a follows the same pattern as the other three histone components of the nucleosome core. Our electron microscope observations have shown that SV40 replicative intermediates indeed contain nucleosomes on prereplicative and on both daughter double helices (unpublished observations). These findings are in agreement with the observations of McKnight and Miller (1977) on Drosophila melanogaster embryo chromatin. To determine whether newly synthesized histones preferentially exchange with the bulk of the histones of the cellular chromatin that comprises the majority of unlabeled histones in the infected cell, we solubilized this fraction and measured the concentration of 35S histones and 3H-DNA. Since the viral infection stimulates cellular DNA synthesis, it is obvious that labeled histones are found
1
FRACTION NUMBER -----1
II
Figure 1. Sedimentation cleoprotein Complexes
UIEP
Analysis
PI
of Mature
and
Replicative
Nu-
(a) CVI cells were infected with a multiplicity of 50 PFU per cell. 41 hr post-infection, the cultures were incubated for 20 min with fresh medium lacking methionine, followed by labeling for 10 min with 1 mCi 55S-methionine (580 Cilmmole) and 0.5 mCi (45 Ci/mmole) of H3-thymidine in 0.5 ml of methionine-free medium. The cells were then washed twice with cold PBS, and the SV40 minichromosomes were extracted and purified by centrifugation in an SW41 rotor at 40,000 rpm for 110 min. Fractions were collected, and aliquots were precipitated and counted. The complexes from different positions of the gradients I-VI as indicated in the figure were pooled, and the complex was pelleted by centrifugation for 14 hr at 50,000 rpm (O0). (b) CVI cells were infected as above and labeled between 32-41 hr post-infection with 100 &i of 35S-methionine and 100 &i of 3H-thymidine in a medium containing l/50 of the normal methionine concentration (x-x).
Figure
2. Analysis
of Histones
Bound
to Mature
and Replicating
DNA
(A and B) SDS-polyacrylamide gel. The complex fractions concentrated by sedimentation (Figure 1) were dissolved in sample buffer and electrophoresed in 17.5% polyacrylamide discontinuous gels according to Blatter et al. (1972). Gels were stained with Coomassie blue, photographed, dipped in DMSO containing PPO and autoradiographed as described by Bonner and Laskey (1974) and Laskey and Mills (1975). (A) stained gel; (B) autoradiogram. (a) histone Hi, sections I-VI from Figure 1; (b) total calf thymus histones; (c) calf thymus histones H3, H2b, H2a and H4; (d) polyoma virus labeled with C’* amino acids. (C and D) Acidic urea polyacrylamide gels. The complex fractions were treated with a buffer containing IO M urea, 0.9 M acetic acid, 5 mg/ml protamine and 1% (w/v) p-mercaptoethanol for 18 hr at 20°C (Schaffhausen and Benjamin, 1976). The samples were separated on 14 cm, 15% gels containing 0.9 M acetic acid and 2.5 M urea as described by Panyim and Chalkley (1969). (C) stained gel; (D) autoradiogram. (a) CVl chromatin, sections I-VI from Figure 1; (b) CVl chromatin and calf thymus histones; (c) histone Hl; (d) calf thymus histones H3, H2b. H2a; (e) calf thymus histones.
Histone 949
a
Association
I II
I
II
with
Replicating
DNA
III
IV
v
VI
Ill
IV
v
VI
b c d e
a
i ii
iii
IV
v
vl’b
c d
Cell 950
Table 1. Histone Gradient
Concentration
in the Different
Sections
of the
Section
I
II
III
IV
Autoradiography (Arbitrary Units)
30
75
69
44
Units)
5
17
53
61
Autoradiography Staining
6
Staining (Arbitrary Ratio
4.4
1 .3
SDS gels (staining and autoradiography) were scanned, the intensitv of bands is expressed in arbitrarv units.
0.72 and
associated with the cellular chromatin. If newly synthesized histones incorporate preferentially into the cellular chromatin, however, we expect that the major fraction of %-methionine-labeled histones are associated with the cellular chromatin, and that the ratio of 35S/3H in the chromatin pellet greatly exceeds that of the viral replicating chromosomes. When measured, this ratio was roughly equal to that of replicating viral chromosomes, excluding the preferential exchange with nonreplieating cellular chromatin. The experiments described in this work analyze only the newly synthesized histones and are confined to a very short time lapse in the viral growth cycle; the cells were pulse-labeled for periods shorter than one viral DNA replication round. We cannot exclude a slow exchange of histones between cellular and viral chromatins occurring throughout the viral growth cycle. In fact, studies on histones associated with polyoma virus showed that a small fraction of cellular histones labeled before the infection are found associated with mature virions (Frearson and Crawford, 1972; Fey and Hirt, 1974). These experiments analyze the entire pool of the viral DNA synthesized over a time span of 2-6 days. At least two hypotheses can be advanced to explain these findings. First, the incorporation of prelabeled cellular histones into viral DNA may involve some exchange of viral and cellular parental histone cores due to a close “functional” association between cellular and viral replicons in the nuclei (LeBlanc and Singer, 1974). Second, during transcription and replication of cellular and viral chromatin, some nucleosomes dissociate and permit a slow histone exchange over a long time span compared to the viral DNA replication cycle. Our results on the preferential association of newly synthesized histones and DNA are in contradiction with the findings of Jackson et al. (1975, 1976) and Seale (1976). who found by density labeling that there was a random distribution of newly synthesized histones. These investigators, however, made several assumptions in the interpretation of their results that were not fully proven.
The possibility that the mechanism of cellular and viral chromatin replication differs extensively seems to us improbable, since the virus utilizes mainly the cellular replication system and coordinately stimulates cellular DNA and histone synthesis. Experlmental
Procedures
Cells and Virus Intaction African green monkey kidney cells (line CVI) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum in 88 mm plastic dishes. Plaque-purified SV40 was used to infect the cell cultures at an input multiplicity of 3050 PFU per cell. At the end of a 2 hr adsorption period, 10 ml of Dulbecco’s modified Eagle’s medium containing 1% serum were added to each dish. Purifkatbn of Nucleoprotein Complexes At 41 hr post-infection, the monolayer was washed twice with phosphate buffer (PBS), the nuclei were isolated in the presence of 0.5% NP40 and the nucleoprotein complex was extracted with 0.2% Triton and 0.2 M NaCl (Green, Miller and Hendler, 1971). Two successive extractions were performed for 30 min at 20°C. The Triton supernatant was obtained after centrifugation at 800 x g, and the nucleoprotein complexes were partially purified by zonal sedimentation on 5-20% (w:w) sucrose gradients in 0.01 M Tris-HCI (pH 7.9), 0.061 M EDTA, 0.2 M NaCl. Gel Electrophoresls Samples from sucrose gradients were concentrated by high speed sedimentation and prepared for analysis of histones. Two different 14 cm long slab gels were used: 17.5% polyacrylamide (Blatter et al., 1972) and acidic urea 15% polyacrylamide gels (Panyim and Chalkley. 1969). In this case, complex fractions were treated with a buffer containing 10 M urea, 0.9 M acetic acid, 5 mg/ml protamine sulfate and 1% (w/v) &mercaptoethanol for 16 hr at 20°C (Schaffhausen and Benjamin, 1978). SDS gels were stained for 1 hr with Coomassie blue and the acidic urea gel with amido black for at least 5 hr. For autoradiography. the gels were impregnated with PPO (Bonner and Laskey. 1974; Laskey and Mills, 1975), dried and clamped to hypersensitized film for exposure at -70°C. Acknowledgments This work was supported by grants from the CNRS, INSERM and the Fondation pour la Recherche Medicale Francaise. We are indebted to Ddile Croissant for providing us with electron microscope facilities, C. Maczuka for help in preparing the manuscript and G. Butler-Browne for valuable criticisms of the manuscript. 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 18 U.S.C. Section 1734 solely to indicate this fact. Received
July 18, 1977;
revised
August
22, 1977
References Blatter, D. P.. Garner, F.. Van Slyke, Chromatography 64, 147-151. Bonner, 88.
M. W. and Laskey.
R. A. (1974).
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