- Email: [email protected]
Inhibition of DNA replication and its effect on histone synthesis
W. G. Flamm and 111.L. Birnstiel
616
an increase in one fraction is paralleled by a similar increase in the other. The maintenance of such an equilibrium is undoubtedly related to the permeability of granules to the enzyme molecules. Changes in granule permeability (as well as in the adsorption of enzyme to granules) can occur during the process of preparation for assay; it is therefore not possible to assess the importance of this phenomenon in development. REFERENCES BLACK, R. E., Biol. Bull. 123, 71 (1962). BRACHET, J., The Biochemistry of Development. Pergamon Press, New York, 1960. GUSTAFSON, T. and HASSELBERG, I., Exptl Cell Res. 2, 642 (1951). MAHLER, H. R., WITTENBERGER, M. H. and BRAND, L., J. Biol. Chem. 233, 770 (1958). SIZER, I. W. and JENKINS, W. T., Methods in Enzymology, Vol. 5, p. 677. S. P. COLOWICK and N. 0. KAPLAN (eds.). Academic Press Inc., New York, 1962. 6. SLATER, E. C. and BONNER, W. D., Biochem. J. 52, 185 (1952). 7. WEBER, R. and BOELL, E. J., Dev. Biol. 4, 452 (1962).
1. 2. 3. 4. 5.
INHIBITION AND
OF DNA
REPLICATION
ITS EFFECT
ON HISTONE
W. G. FLAMM
and M. L. BIRNSTIELI
Division of Biology, California
SYNTHESIS
Institute of Technology, Pasadena, Calif., U.S.A.
Received December 9, 1963
Tm localization
of histones in the nucleus and their strong interaction with DNA suggests the possibility that they may play an important role in the organization of DNA into the superstructure of the chromosome. Studies by Bonner and Huang [4, 91 further suggest that the histone component of the chromosome exerts control over genetic activity. That is, histones may, by complexing with DNA and thereby inhibiting DNA directed RNA synthesis, prevent the transcription of genetic information. In view of this suggested role of histones in chromosome activity, it is of interest to know whether or not there is any relationship between the synthesis of histone and that of DNA. Our approach to this study was to selectively inhibit the replication of DNA in exponentially multiplying cells and to determine the effect of this inhibition on histone synthesis. Materials and methods.-Tobacco cells, derived from the stem of Nicotiana tabacum variety Xanthi were grown and subcultured in liquid medium as earlier described [7]. Only those cells which had been subcultured at least 30 times were employed in these experiments and all cells, except where otherwise noted, were multiplying exponentially when used. 1 Present address: Institute
of Animal
Experimental Cell Research 33
Genetics, University
of Edinburgh,
Edinburgh,
Scotland.
DNA replication
and histone synthesis
617
5-Fluorodeoxyuridine was added directly to the cell culture media to attain the concentrations specified in Fig. 2 and Table I. The cultures were allowed to incubate under otherwise normal growth conditions for various lengths of time in the appropriate concentration of 5-fluorodeoxyuridine after which the cells were harvested by filtration through Miracloth. For each incubation of cells with radioactive precursors, 20 to 40 g (fresh weight) of filtered cells were suspended in 25 to 50 ml of
eoyr ~CulfureAgel
-Log I 5 Fl”orodeory”ridi”e,
Fig. 1.
Fig. 2.
Fig. l.-The relative rate of histone synthesis and the fresh weight increase of cells as functions of culture age. Incubation conditions and fractionations as specified in the text. acid into RNA and Fig. 2.-Effect of 5-fluorodeoxyuridine upon the conversion of %-erotic DNA. Cell cultures were incubated in the presence of 5-fluorodeoxyuridine for 2 hr, then harvested and incubated with 20 pc W-erotic acid for 1 hr as specified in the text.
growth medium containing the specified concentration of 5-fluorodeoxyuridine and incubated with either 100 ,LLC of 14C algal protein hydrolysate (0.6 mg/mC) or 20 ,uc of 14C-2-erotic acid. Incorporation of labeled precursors was terminated by the addition of 10 volumes of ice-cold grinding medium (0.01 LWtris-HCl, pH 7.5; 0.025 M sucrose) and by immediate filtration of the cells at 0”. Nuclei were isolated and purified by centrifugation through a dextran gradient (18 to 2 per cent) as earlier described [7]. The nuclear preparations thus obtained were fractionated by previously described procedures [6] to yield the 5 protein fractions listed in Table I. This involved the sequential extraction of ribonucleoprotein particles with 0.5 per cent desoxycholate followed by the extraction of basic proteins with 0.2 N HCl (90”; 30 min). The basic proteins were then precipitated with an equal volume of 40 per cent TCA and further fractionated into histone and non-histone proteins by extraction of the former with the Mirsky-Pollister reagent [ll] as earlier described [l]. Nucleic acid and protein analyses were conducted according to the procedures of Experimental
Cell Research 33
W. G. Flamm and M. L. Birnstiel
618
Schmidt ef al. [12] and Lowry et al. [lo], respectively. Radioactivity of extracted protein and nucleic acid fractions was determined by methods standard to this laboratory [2, 51. Results.-The
proportion
of histones synthesized
relative
to the total synthesis
of
nuclear proteins is maximal during the exponential growth phase and falls off sharply when the rate of cell replication decreases (Fig. 1). These observations are TABLE
I. Effect of 5-fluorodeoxyuridine
upon nuclear protein synthesis.
Tobacco cells were pretreated with lo-? M 5-fluorodeoxyuridine under growth conditions for varying lengths of time. The cells were collected and incubated for one hour in 50 ml of growth medium containing 100 ,uc % algal protein hydrolysate and lo-’ A4 5-fluorodeoxyuridine. Specific activity of protein as per cent of control’ Hours pretreatment with 5-FDU Fraction Histone Basic non-hi&one Residual Ribonucleoprotein Supernatant
0.5
3
6
18
84 97 96 89 112
89 91 89 86 109
96 103 110 109 99
102 122 134 115 110
a Not 5-FDU treated.
quite similar to those of Bloch [3] who also found that DNA synthesis is normally accompanied by increased histone production. The possibility that histone synthesis is dependent upon DNA or cell replication was also considered by Bloch and it was further suggested that the syntheses of these two substances might be inextricably coupled. To test this hypothesis we have worked out conditions under which DNA and cell replication may be blocked without affecting the initial rate of RNA synthesis as measured by the conversion of 14C-erotic acid into RNA. The data of Fig. 2 show that
concentrations
of 5-fluorodeoxyuridine
of lo-’
to 1O-6 M effectively
inhibit
the conversion of erotic acid into DNA while the rate of incorporation of erotic acid into RNA is not affected. Although the data of Fig. 2 represent only those results obtained after a two hour treatment of cells with 5-fluorodeoxyuridine, we have obtained similar inhibition patterns for treatment times ranging from 0 to 18 hr. Furthermore, microscopic examination of 5-fluorodeoxyuridine-treated cells revealed the absence of mitotic figures which were readily observable in the untreated control cells. That the treated cells remain viable and that the effect of 5-fluorodeoxyuridine is restricted to inhibition of thymidylate synthetase [8] and hence to its effect on cellular metabolism is indicated by the fact that the addition of thymidine brought about the rapid resumption of both DNA and cell replication. As can be seen from the data of Table I, the inhibition of DNA synthesis and of mitosis does not significantly alter, over the time period studied, the rate of amino Experimental
Cell Pesearch 33
DNA
replication
and histone synthesis
619
acid incorporation into any of the five nuclear protein fractions studied including the histone fraction. That our amino acid incorporation data actually reflect rate of protein synthesis is indicated by the results of pulse-chase experiments in which 5-fluorodeoxyuridine-treated and control cells, incubated for one hour in 14Camino acids, were treated with a lOOO-fold excess of nonradioactive amino acids. It was found that the changes in specific activity of the nuclear proteins during the chase portion of the experiment were qualitatively and quantitatively the same in both treated and control cells. In addition it was observed that the total nuclear protein of 5-fluorodeoxyuridine-treated cells increased by 30 per cent over an 18 hr period. It is clear, therefore, that histone synthesis and accumulation can continue in the absence of DNA or cell replication. The possibility that DNA replication depends upon histone synthesis, however, has not been eliminated. The authors wish to thank Professor James Bonner for his many helpful suggestions and interest during the course of this work. This investigation was supported by a grant (GM-03977) from the U.S. Public Health Service. The first author is a Postdoctoral Fellow of the U.S. Public Health Service. REFERENCES 1. BIRNSTIEL, M. L. and FLAMM, W. G., Biochim. Biophys. Acta (In press). 2. BIRNSTIEL; M. L. and HYDE,.B. B., j. Cell Biol. Is, 41 (1963): ’ 3. BLOCH, D. P. and GODMAN, G. C., J. Biophys. Biochem. Cytol. 1, 17 (1955). 4. BONNER, J., HUANG, R. C. and GILDEN, R. V., Proc. Natl. Acad. Sci. U.S. 50, 893 (1963). 5. FLAMM, W. G. and BIRNSTIEL, M. L., in Biology and Chemistry of Histones, p. 480. J. BONNER and P. 0. P. Ts’o (eds.) Holden-Day, Inc., San Francisco, 1963. 6. Biochim. Biophys. Acta (In press). 7. FLAMM, W. G., BIRNSTIEL, M. L. and FILNER, P., Biochim. Biophys. Acta 76, 110 (1963). 8. HARBERS, E., CHAUDHLJRI, N. K. and HEIDELBERGER, C., J. Biol. Chem. 234, 1255 (1959). 9. HUANG, R. C: and BONN&, J., Proc. Natl Acad. Sci. U.S. 48, 1216 (1962). 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J., .I. Biol. Chem. 193, 265 (1951). 11. MIRSKY, A. E. and POLLISTER, A. W., J. Gen. Physiol. 30, 117 (1946). 12. SCHMIDT, G. and THANNHAUSER, S. J., J. Biol. Chem. 161, 83 (1945).
Experimental
Cell Research 33
616
an increase in one fraction is paralleled by a similar increase in the other. The maintenance of such an equilibrium is undoubtedly related to the permeability of granules to the enzyme molecules. Changes in granule permeability (as well as in the adsorption of enzyme to granules) can occur during the process of preparation for assay; it is therefore not possible to assess the importance of this phenomenon in development. REFERENCES BLACK, R. E., Biol. Bull. 123, 71 (1962). BRACHET, J., The Biochemistry of Development. Pergamon Press, New York, 1960. GUSTAFSON, T. and HASSELBERG, I., Exptl Cell Res. 2, 642 (1951). MAHLER, H. R., WITTENBERGER, M. H. and BRAND, L., J. Biol. Chem. 233, 770 (1958). SIZER, I. W. and JENKINS, W. T., Methods in Enzymology, Vol. 5, p. 677. S. P. COLOWICK and N. 0. KAPLAN (eds.). Academic Press Inc., New York, 1962. 6. SLATER, E. C. and BONNER, W. D., Biochem. J. 52, 185 (1952). 7. WEBER, R. and BOELL, E. J., Dev. Biol. 4, 452 (1962).
1. 2. 3. 4. 5.
INHIBITION AND
OF DNA
REPLICATION
ITS EFFECT
ON HISTONE
W. G. FLAMM
and M. L. BIRNSTIELI
Division of Biology, California
SYNTHESIS
Institute of Technology, Pasadena, Calif., U.S.A.
Received December 9, 1963
Tm localization
of histones in the nucleus and their strong interaction with DNA suggests the possibility that they may play an important role in the organization of DNA into the superstructure of the chromosome. Studies by Bonner and Huang [4, 91 further suggest that the histone component of the chromosome exerts control over genetic activity. That is, histones may, by complexing with DNA and thereby inhibiting DNA directed RNA synthesis, prevent the transcription of genetic information. In view of this suggested role of histones in chromosome activity, it is of interest to know whether or not there is any relationship between the synthesis of histone and that of DNA. Our approach to this study was to selectively inhibit the replication of DNA in exponentially multiplying cells and to determine the effect of this inhibition on histone synthesis. Materials and methods.-Tobacco cells, derived from the stem of Nicotiana tabacum variety Xanthi were grown and subcultured in liquid medium as earlier described [7]. Only those cells which had been subcultured at least 30 times were employed in these experiments and all cells, except where otherwise noted, were multiplying exponentially when used. 1 Present address: Institute
of Animal
Experimental Cell Research 33
Genetics, University
of Edinburgh,
Edinburgh,
Scotland.
DNA replication
and histone synthesis
617
5-Fluorodeoxyuridine was added directly to the cell culture media to attain the concentrations specified in Fig. 2 and Table I. The cultures were allowed to incubate under otherwise normal growth conditions for various lengths of time in the appropriate concentration of 5-fluorodeoxyuridine after which the cells were harvested by filtration through Miracloth. For each incubation of cells with radioactive precursors, 20 to 40 g (fresh weight) of filtered cells were suspended in 25 to 50 ml of
eoyr ~CulfureAgel
-Log I 5 Fl”orodeory”ridi”e,
Fig. 1.
Fig. 2.
Fig. l.-The relative rate of histone synthesis and the fresh weight increase of cells as functions of culture age. Incubation conditions and fractionations as specified in the text. acid into RNA and Fig. 2.-Effect of 5-fluorodeoxyuridine upon the conversion of %-erotic DNA. Cell cultures were incubated in the presence of 5-fluorodeoxyuridine for 2 hr, then harvested and incubated with 20 pc W-erotic acid for 1 hr as specified in the text.
growth medium containing the specified concentration of 5-fluorodeoxyuridine and incubated with either 100 ,LLC of 14C algal protein hydrolysate (0.6 mg/mC) or 20 ,uc of 14C-2-erotic acid. Incorporation of labeled precursors was terminated by the addition of 10 volumes of ice-cold grinding medium (0.01 LWtris-HCl, pH 7.5; 0.025 M sucrose) and by immediate filtration of the cells at 0”. Nuclei were isolated and purified by centrifugation through a dextran gradient (18 to 2 per cent) as earlier described [7]. The nuclear preparations thus obtained were fractionated by previously described procedures [6] to yield the 5 protein fractions listed in Table I. This involved the sequential extraction of ribonucleoprotein particles with 0.5 per cent desoxycholate followed by the extraction of basic proteins with 0.2 N HCl (90”; 30 min). The basic proteins were then precipitated with an equal volume of 40 per cent TCA and further fractionated into histone and non-histone proteins by extraction of the former with the Mirsky-Pollister reagent [ll] as earlier described [l]. Nucleic acid and protein analyses were conducted according to the procedures of Experimental
Cell Research 33
W. G. Flamm and M. L. Birnstiel
618
Schmidt ef al. [12] and Lowry et al. [lo], respectively. Radioactivity of extracted protein and nucleic acid fractions was determined by methods standard to this laboratory [2, 51. Results.-The
proportion
of histones synthesized
relative
to the total synthesis
of
nuclear proteins is maximal during the exponential growth phase and falls off sharply when the rate of cell replication decreases (Fig. 1). These observations are TABLE
I. Effect of 5-fluorodeoxyuridine
upon nuclear protein synthesis.
Tobacco cells were pretreated with lo-? M 5-fluorodeoxyuridine under growth conditions for varying lengths of time. The cells were collected and incubated for one hour in 50 ml of growth medium containing 100 ,uc % algal protein hydrolysate and lo-’ A4 5-fluorodeoxyuridine. Specific activity of protein as per cent of control’ Hours pretreatment with 5-FDU Fraction Histone Basic non-hi&one Residual Ribonucleoprotein Supernatant
0.5
3
6
18
84 97 96 89 112
89 91 89 86 109
96 103 110 109 99
102 122 134 115 110
a Not 5-FDU treated.
quite similar to those of Bloch [3] who also found that DNA synthesis is normally accompanied by increased histone production. The possibility that histone synthesis is dependent upon DNA or cell replication was also considered by Bloch and it was further suggested that the syntheses of these two substances might be inextricably coupled. To test this hypothesis we have worked out conditions under which DNA and cell replication may be blocked without affecting the initial rate of RNA synthesis as measured by the conversion of 14C-erotic acid into RNA. The data of Fig. 2 show that
concentrations
of 5-fluorodeoxyuridine
of lo-’
to 1O-6 M effectively
inhibit
the conversion of erotic acid into DNA while the rate of incorporation of erotic acid into RNA is not affected. Although the data of Fig. 2 represent only those results obtained after a two hour treatment of cells with 5-fluorodeoxyuridine, we have obtained similar inhibition patterns for treatment times ranging from 0 to 18 hr. Furthermore, microscopic examination of 5-fluorodeoxyuridine-treated cells revealed the absence of mitotic figures which were readily observable in the untreated control cells. That the treated cells remain viable and that the effect of 5-fluorodeoxyuridine is restricted to inhibition of thymidylate synthetase [8] and hence to its effect on cellular metabolism is indicated by the fact that the addition of thymidine brought about the rapid resumption of both DNA and cell replication. As can be seen from the data of Table I, the inhibition of DNA synthesis and of mitosis does not significantly alter, over the time period studied, the rate of amino Experimental
Cell Pesearch 33
DNA
replication
and histone synthesis
619
acid incorporation into any of the five nuclear protein fractions studied including the histone fraction. That our amino acid incorporation data actually reflect rate of protein synthesis is indicated by the results of pulse-chase experiments in which 5-fluorodeoxyuridine-treated and control cells, incubated for one hour in 14Camino acids, were treated with a lOOO-fold excess of nonradioactive amino acids. It was found that the changes in specific activity of the nuclear proteins during the chase portion of the experiment were qualitatively and quantitatively the same in both treated and control cells. In addition it was observed that the total nuclear protein of 5-fluorodeoxyuridine-treated cells increased by 30 per cent over an 18 hr period. It is clear, therefore, that histone synthesis and accumulation can continue in the absence of DNA or cell replication. The possibility that DNA replication depends upon histone synthesis, however, has not been eliminated. The authors wish to thank Professor James Bonner for his many helpful suggestions and interest during the course of this work. This investigation was supported by a grant (GM-03977) from the U.S. Public Health Service. The first author is a Postdoctoral Fellow of the U.S. Public Health Service. REFERENCES 1. BIRNSTIEL, M. L. and FLAMM, W. G., Biochim. Biophys. Acta (In press). 2. BIRNSTIEL; M. L. and HYDE,.B. B., j. Cell Biol. Is, 41 (1963): ’ 3. BLOCH, D. P. and GODMAN, G. C., J. Biophys. Biochem. Cytol. 1, 17 (1955). 4. BONNER, J., HUANG, R. C. and GILDEN, R. V., Proc. Natl. Acad. Sci. U.S. 50, 893 (1963). 5. FLAMM, W. G. and BIRNSTIEL, M. L., in Biology and Chemistry of Histones, p. 480. J. BONNER and P. 0. P. Ts’o (eds.) Holden-Day, Inc., San Francisco, 1963. 6. Biochim. Biophys. Acta (In press). 7. FLAMM, W. G., BIRNSTIEL, M. L. and FILNER, P., Biochim. Biophys. Acta 76, 110 (1963). 8. HARBERS, E., CHAUDHLJRI, N. K. and HEIDELBERGER, C., J. Biol. Chem. 234, 1255 (1959). 9. HUANG, R. C: and BONN&, J., Proc. Natl Acad. Sci. U.S. 48, 1216 (1962). 10. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J., .I. Biol. Chem. 193, 265 (1951). 11. MIRSKY, A. E. and POLLISTER, A. W., J. Gen. Physiol. 30, 117 (1946). 12. SCHMIDT, G. and THANNHAUSER, S. J., J. Biol. Chem. 161, 83 (1945).
Experimental
Cell Research 33