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Incorporation of radioisotope, in vivo, into ribonucleic acid and histone of a fraction of nuclei preparing for mitosis
Experimenfal
INCORPORATION INTO
435
Cell Research 39, 435-442 (1965)
RIBONUCLEIC OF NUCLEI
OF RADIOISOTOPE, ACID AND
Department
HISTONE
PREPARING
W. G. NIEHAUS,
JR?
IN VIVO, OF A FRACTION
FOR MITOSIS1
and C. P. BARNUM
of Biochemistry, University of Minnesota Minneapolis, Minn., U.S.A.
Medical
School,
Received January 5, 1965
THE synthesis of DNA is known to occur during the late part of interphase in vertebrate cells [al]. On this basis, the cell cycle may be divided into four discrete phases: G1, the postmitotic period preceding DNA synthesis; period following DNA S, the period of DNA synthesis; G2, the premitotic synthesis; and M, the relatively brief period of mitotic division [14]. In regenerating rat liver, the duration of the S and G2 phases is about 8 hr each [IS; 171. A method for the isolation from regenerating rat liver, of nuclei which have increased their content of DNA prior to mitosis has been described previously [lo]. Hepatocyte nuclei are separated by density gradient centrifugation into a fraction with the tetraploid content of DNA characteristic of adult rat liver, and a more dense fraction of nuclei with a content of DNA approaching the octaploid condition [lo]. After 4 hr of incorporation of isotope in uiuo, the DNA of the more dense fraction of nuclei has a higher specific activity than the DNA of the lighter fraction [19]. It has been concluded [lo, 191 that the nuclei of the more dense fraction have increased their content of DNA prior to mitosis, and therefore may be considered to be in the G, phase or in the late part of the S phase of the cell cycle. The less dense nuclei then may be considered to be in the G1 phase or in the early part of the S phase of the cell cycle.
1 This investigation was supported by Public Health Service Research Grant No. CA-01747-13 from the National Cancer Institute and by Grant No. IN-13B from the American Cancer Society. * Predoctoral support provided by the United States Public Health Service Medical Student Research Training Grant MSRT-2R-24 and by Graduate Training Grant 2G-5Tl-GM-157-05 from the National Institutes of Health, United States Public Health Service. The data presented were taken from a thesis submitted to the Faculty of the Graduate School, University of Minnesota, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois. Experimental
Cell Research 39
436
W. G. Niehaus, Jr. and C. P. Barnum
It has been reported that RNA synthesis is depressed during the period of DNA synthesis (S phase) in Tetrahymena [22], in root tip nuclei [25], in the slime mold [20], and in Euplotes [23], as well as in cultured mammalian cells [24] and in regenerating rat liver nuclei [27]. Nuclear histone, however, appears to be synthesized during the S phase in root tip nuclei [l] and in Euplotes [ll, 231. Experiments have been performed to investigate the metabolism of RNA and histone during the S and G, phases of the cell cycle. The uptake of isotope, in vivo, into RNA and histone has been compared in the two fractions of nuclei, one fraction representing the G, and late part of the S phase, the other representing the G, and the early part of the S phase.
EXPERIMENTAL
PROCEDURE
Male Holtzman rats weighing 250 to 400 g were used in these experiments. Partial hepatectomies were performed by the method of Higgins and Anderson [12] as modified by Brues, Drury and Brues [6]. All partial hepatectomies were performed about 8 a.m. At specified times after partial hepatectomy, 32P-orthophosphate (carrier-free, Nuclear Consultants Corporation) at a level of 1 ,LLC per g of body weight, or 2-W-glycine (Tracerlab) at a level of 0.1 PC per g of body weight, was administered as a single intraperitoneal injection. At various times after administration of isotope the animals were killed and the livers removed. All isolation procedures were performed in a cold room at 4”. The liver was homogenized in a smooth glass homogenizer with a Teflon pestle in 9 volumes of cold 0.25 M sucrose that was 5 mM with respect to CaCl,. In those experiments in which histone was to be isolated, the sucrose solutions were adjusted to pH 5.8 with 0.12 M acetate buffer to minimize proteolysis during the isolation procedure [9]. In the experiments employing snP, a portion of the homogenate was removed for the isolation of acid-soluble nucleotides. Nuclei were isolated from the homogenate by a slight modification of the method of Fisher, Holbrook and Irvin [lo], as described previously [19]. Upon centrifugation through a sucrose density gradient the nuclei were separated into a pellet plus three bands of nuclei. The pellet fraction contains the premitotic nuclei in G, and late S phases, and the lower layer contains the hepatocyte nuclei in G, and early S phases. The middle and upper layers, which contain non-parenchymal nuclei [lo], were not used in these experiments. Preparation
of RNA, acid-soluble
nucleotides, and proteins
In the experiments employing azP, the nucleic acids were extracted from the isolated nuclei and the RNA hydrolyzed and ashed as described previously [19]. Acidsoluble nucleotides were adsorbed onto washed charcoal from the supernatant fluid from the trichloracetic acid precipitation of the homogenate. The charcoal containing the absorbed nucleotides was washed once with distilled water and the nucleotides Experimental
Cell Research 39
Metabolism
of RNA and histone in nuclei prior to mitosis
437
were eluted with a mixture of ethanol, water and concentrated ammonia (50:45:5) and ashed. The specific activities of RNA and acid-soluble nucleotides were determined as described [19]. In those experiments in which histone was to be isolated, the nuclei were suspended in 0.25 N H,SO, and stirred continuously at 4” for 2 hr, then centrifuged 10 min at 600 xg. The pellet, containing nucleic acid and acid-insoluble nuclear protein, was heated with buffered 10 per cent NaCl to solubilize the nucleic acids as described 1191.The nucleic acids were precipitated from the 10 per cent NaCl with two volumes of ethanol and the RNA was removed by alkaline hydrolysis. The DNA was hydrolyzed in 5 per cent trichloroacetic acid and the hydrolysate adsorbed onto charcoal and eluted 1261. Histone was purified by a modification of the method of Laurence, Simson and Butler [15]. The histones were precipitated from the 0.25 N H,SO, by the addition of trichloroacetic acid to a final concentration of 20 per cent. The histone was redissolved in 0.25 N H,SOI by stirring at room temperature for I hr. Some of the protein did not return to solution in this step and was discarded. The histone was again precipitated by addition of trichloroacetic acid to 20 per cent concentration, and the precipitate was redissolved and precipitated. The precipitate was then washed with acetone, dried, and dissolved in 0.004 N HCl. DNA and histone samples were plated directly onto aluminium planchets at infinite thinness, dried and counted on a windowless gas flow counter. DNA was measured by the diphenylamine method of Burton [7] and histone was measured by the method of Lowry, Rosebrougb, Farr and Randall [18], using commercial histone (Worthington) as a standard. RESULTS
Incorporation
of 32P into RNA
When 32P, was administered 18.5 or 27.5 hr after partial hepatectomy (at times near the onset of DNA synthesis and near its peak) and the animals killed 30 min later, the RNA from nuclei in the pellet fraction had a specific activity about 1.5 times that of the RNA from the lower layer of nuclei (Table I). The possibility was considered that the differences in RNA specific activity between the two fractions of nuclei were due to contamination of the nuclei from the lower layer by cytoplasmic RNA, which has a much lower specific activity than does nuclear RNA. Nuclei from the lower layer and from the pellet were isolated and treated with citric acid [3]. As shown in Table II, no significant differences were found between citric acid-treated and untreated nuclei from each fraction. Incorporation
of 14C into DNA and histone
When 2-14C-glycine was administered 26 hr after partial hepatectomy and the animals were killed 2 hr later, the specific activity of histone from the 28 -
651816
Experimental
Cell Research 39
438
W. G. Niehaus, Jr. and C. P. Barnum
pellet fraction of nuclei was 1.6 times that from the lower layer of nuclei, while the ratio for DNA was 2.1. This result with DNA confirmed earlier work using 32Pi as an isotopic precursor and allowed the comparison of DNA and histone in the same animal. When isotope was administered 17 hr TABLE
I. Comparison
of specific activities
of RNA from two fractions
sePi, 1 ,uc per g of body weight, was injected at the indicated later. Each line represents the pooled livers of two rats. Time of isotope administration (hr after partial hepatectomy) 18.5 18.5 18.5 27.5 27.5 27.5 27.5 27.5 27.5
Specific activity (per cent of acid-soluble nucleotide activity) r-h-7 Pellet Lower 26 25 26 18 20 25 17 16 14
37 36 40 23 27 37 27 28 29
time. Animals
of nuclei.
were killed
30 min
Ratio of specific activity Pellet/Lower 1.4 1.4 1.5 1.3 1.3 1.5 1.6 1.7 2.1
after partial hepatectomy and the animals were killed 2 hr later, the specific activity of histone from the pellet fraction was also 1.6 times that from the lower layer of nuclei (Table III).
IHSCUSSION
Incorporation
of 32P into RNA
As shown in Tables I and II, the specific activity of RNA from the pellet fraction of nuclei is greater than that of RNA from the lower nuclear layer, when isotope is administered at two different times after partial hepatectomy. Data in Table II have been interpreted as demonstrating that the smaller RNA specific activity in the lower nuclear layer is not due to contamination by cytoplasmic RNA. Citric acid treatment in the process of isolation of nuclei is known to remove nearly all cytoplasmic contamination, resulting in a progressive increase in the specific activity of the RNA of the nuclear Experimental
Cell Research 39
Metabolism
of RNA and histone in nuclei prior to mitosis
439
preparation [a]. Since no increase in RNA specific activity was seen in these isolated nuclei following subsequent citric acid treatment, it was concluded that the samples of nuclei were not contaminated by cytoplasmic RNA. In order to compare the rates of RNA synthesis at the two time points TABLE
II. Comparison of specific activities of RNA from two fractions before and after citric acid treatment.
asPi, 1 pc per g of bodv weight, was injected 27.5 hr after partial hepatectomy. killed 30 min later. Each line represents the pooled livers of four rats. Specific activity (per cent of acid-soluble nucleotide activity) Lower before citric acid 13 20 20
Lower after citric acid 14 20 17
Pellet before citric acid 17 26
Ratio Pellet after citric acid 16 26 -
Lower after citric acid/ Lower before citric acid 1.1 1.0 0.9
of nuclei
Animals
were
of specific activity Pellet after citric acid/ Pellet before citric acid 0.9 1.0 .-
Pellet/ Lower (untreated nuclei) 1.3 1.3 -
employed, RNA specific activity was calculated as a per cent of the specific activity of acid soluble nucleotide phosphorus, which is presumed to be representative of the precursor pool. The results are consistent with the finding of Welling and Cohen [27] that nuclear RNA synthesis is more rapid 19 hr after partial hepatectomy than after 28 hr. The pellet fraction of nuclei is proposed to consist of two classes of nuclei; those which are in the process of DNA synthesis (S phase), and those which have completed DNA synthesis prior to mitosis (G, phase) [19]. Therefore, the increased RNA specific activity in the pellet fraction over that of the lower layer of nuclei may be due either to a coincident synthesis of RNA and DNA, or to an increase in the rate of RNA synthesis in the period between the end of DNA synthesis and the onset of mitosis (G, phase). As has been mentioned, RNA synthesis is known to be depressed during DNA synthesis in several invertebrate organisms [20, 22, 23, 251. Welling and Cohen [27] have found that in regenerating rat liver, peak synthesis of nuclear RNA occurs about 16 hr after partial hepatectomy and falls off rapidly, reaching a very low level by 24 hr, when DNA synthesis is maximal. Reiter and Littlefield [24], studying cultures of mouse fibroblasts partially synchronized with 5-fluoroExperimental
Cell Research 39
440
W. G. Niehaus, Jr. and C. P. Barnum
deoxyuridine, found the synthesis of nuclear RNA to be depressed somewhat during the period of DNA synthesis, increasing markedly as the latter slowed, and remaining active until the onset of mitosis. Therefore, the more likely interpretation of the data presented in Table I seems to be that the higher TABLE
III.
Comparison
of specific activities of histone fractions of nuclei.
and DNA from
2-‘Gglycine, 0.1 ,uc per g of body weight, was injected at the indicated killed 2 hr later. Each line represents the pooled livers of two rats. Time of isotope administration (hr after partial hepatectomy) 17 17 17 17 26 26 26 26 26 26
time.
Animals
two were
Specific activity DNA (c.p.m./mg DNA) Lower
Pellet
1320 1520 1010 1775
2870 3300 2120 3630
Histone (c.p.m./mg protein) Lower
Pellet
3670 2640 3690 4950 3600 1440 3590 2970 2770 2530
4770 4180 5840 9100 4270 1820 5040 5020 5200
Ratio of specific activity c-A-. DNA Histone Pellet/Lower Pellet/Lower
-
2.2 2.2 2.1 2.0
1.3 1.6 1.6 1.8 1.2 1.3 1.6 1.7 1.8 2.1
RNA specific activity in the pellet fraction of nuclei is due to an increase in nuclear RNA synthesis following the completion of DNA synthesis, i.e., during the G, phase of the cell cycle. Incorporation
of 14C into histone
Reports in the literature indicate that histone and DNA4 are synthesized simultaneously in root tip nuclei [l] and in Euplotes [ll, 231. The time of histone synthesis in mammalian tissues, however, is not clearly defined. Several studies performed using regenerating rat liver have yielded ambiguous results. Holbrook et al. [13] concluded that histone and DNA are synthesized at the same time. Butler and Cohn [S] found the peak of histone synthesis to precede that of DNA. Bloch and Godman [5] originally concluded that histone and DNA are synthesized simultaneously; however, Bloch [4] later reversed this position and stated that a more likely interpretation of his Experimental
Cell Research 39
Metabolism
of RNA and hisfone in nuclei prior
to mitosis
441
earlier work was that histone was synthesized earlier and associated with the DNA immediately after DNA synthesis. There are no indications in the literature that DNA synthesis could precede histone synthesis. Bloch and Godman [5] found newly synthesized DNA complexed with histone even when the nucleus contained an amount of DNA intermediate between diploid and tetraploid. Such an intermediate nucleus is presumed to be in the process of DNA synthesis. Therefore, histone must have been synthesized before or at the time of DNA synthesis. The specific activity of histone was found to be greater in the nuclei of the pellet fraction than in the nuclei of the lower layer after a two-hour period of isotope incorporation in vivo (Table III). It has been proposed that the nuclei in the pellet fraction are in the G, phase or the late part of the S phase of the cell cycle, having increased their content of DNA prior to mitosis. If histone were synthesized earlier than DNA, histone synthesis in these nuclei of the pellet fraction would have been completed before the addition of isotope, and the specific activity of histone in these nuclei would be expected to be low. Since, in fact, the specific activity of histone is found to be higher in these nuclei, it would appear that in the regenerating liver, histone synthesis is increased either during the S phase or the G, phase of the cell cycle. As was mentioned above, it seems unlikely that histone synthesis follows the synthesis of DNA; therefore, the more likely interpretation of the data presented in Table III seems to be that the higher specific activity of histone in the pellet fraction of nuclei is due to an increase in histone synthesis during the period of DNA synthesis, i.e., during the S phase of the cell cycle. Histone as isolated from nuclear fractions by the method of Lawrence et al. [15] would also be expected to contain small amounts of other basic proteins, e.g., those associated with nuclear ribosomes. Therefore the possibility must be considered that the increased incorporation of radioactivity may also reflect an increased synthesis of these basic proteins.
SUMMARY
Following the administration of radioisotope in vivo, nuclei were isolated from regenerating rat liver and separated into two fractions, one of which consisted of nuclei which had increased their DNA content preparatory to mitosis. In this premitotic fraction of nuclei, both RNA and histone were more highly labeled than in the fraction of nuclei with a normal DNA content. Experimental
Cell Research 39
442
W. G. Niehaus, Jr. and C. P. Barnum
It was concluded that histone and DNA are synthesized during the same period, and that there is an increase in the synthesis of nuclear RNA immediately following the cessation of DNA synthesis. REFERENCES 1. ALFERT, M., Exptl Cell Res. Suppl. 6, 227 (1959). C; P.,*Unpublished observations.‘ 2. BARNUM, 3. BARNUM, C. P., NASH. C. W.. JENNINGS, E., NYGAARD, 0. and VERMUND, H., Arch. Bio&m. 25;376 (i950). 4. BLOCH, D. P., J. Cell. Comp. Physiol. 62 Suppl. I, 87 (1963). 5. BLOCH, D. P. and GODMAN, G. C., J. Biophys. Biochem. Cytol. 1, 17 (1955). 6. BRUES, A. M., DRURY, D. R. and BRUES, M. C., Arch. Pathol. 22, 658 (1936). 7. BURTON, K., Biochem. J. 62, 315 (1956). 8. BUTLER, J. A. V. and COHN, P., Biochem. J. 87, 330 (1963). 9. DOUNCE, A. L. and UMANA, R., Biochem. 1, 811 (1962). 10. FISHER, k. F., HOLBROOK, D. J. and IRVIN, J. L:, J. Cell. Biol. 17, 231 (1963). 11. GALL, J. G., J. Biophys. Biochem. Cytol. 5, 295 (1959). G. M. and ANDERSON, R. M., Arch. Pathol. 12, 186 (1931). 12. HIGGINS, D. J., EVANS, J. H. and IRVIN, J. L.. Erptl Cell Res. 28, 120 (1962). 13. HOLBROOK, 14. HOWARD, A. and’PE~c, S: R., Heredity SuppI. 6, ‘261’(1953). D. J. R., SIMSON, P. and BUTLER, J. A. V., Biochem. .7. 87, 200 (1963). 15. LAURENCE, 16. LOONEY, W. B., Proc. Nat1 Acad. Sci. U.S. 46, 690 (1960). in J. S. MITCHELL (ed.), The Cell Nucleus, p. 98. Academic Press, New York, 1960. 17. ~ N. J., FARR, A. L. and RANDALL, R. J., J. Biol. Chem. 193, 18. LOWRY, 0. H., ROSEBROUGH, 265 (1951). W. G. and BARNUM, C. P., J. Biot. Chem. 239, 1198 (1964). 19. NIEHAUS, 20. NYGAARD, 0. F., G~~TTES, S. and RUSCH, H. P., Biochim. Biophys. Acta 38, 298 (1960). 21. PRESCOTT, D. M., Intern. Reu. Cytol. 11, 255 (1961). 22. __ Exptl Cell Res. 19, 228 (1960). R. F.. Proc. Natl Acad. Sci. U.S. 47. 686 (1961). 23. PRESCOTT. D. M. and KIMBALL, 24. REITER, j. M. and LITTLEFIELD, J. w., Biochim. Biophys. Acta 80, 562 (1664).’ 25. SISKEN, J. E., Expfl Cell Res. 16, 602 (1959). 26. Snnm, I. (ed.), Chromatographic and Electrophoretic Techniques, Vol. 1, p. 242. Intersciencc Publishers, New York, 1960. 27. WELLING, W. and COHEN, J. A., Biochim. Biophys. Acta 42, 181 (1960).
Experimental
Cell Research 39
INCORPORATION INTO
435
Cell Research 39, 435-442 (1965)
RIBONUCLEIC OF NUCLEI
OF RADIOISOTOPE, ACID AND
Department
HISTONE
PREPARING
W. G. NIEHAUS,
JR?
IN VIVO, OF A FRACTION
FOR MITOSIS1
and C. P. BARNUM
of Biochemistry, University of Minnesota Minneapolis, Minn., U.S.A.
Medical
School,
Received January 5, 1965
THE synthesis of DNA is known to occur during the late part of interphase in vertebrate cells [al]. On this basis, the cell cycle may be divided into four discrete phases: G1, the postmitotic period preceding DNA synthesis; period following DNA S, the period of DNA synthesis; G2, the premitotic synthesis; and M, the relatively brief period of mitotic division [14]. In regenerating rat liver, the duration of the S and G2 phases is about 8 hr each [IS; 171. A method for the isolation from regenerating rat liver, of nuclei which have increased their content of DNA prior to mitosis has been described previously [lo]. Hepatocyte nuclei are separated by density gradient centrifugation into a fraction with the tetraploid content of DNA characteristic of adult rat liver, and a more dense fraction of nuclei with a content of DNA approaching the octaploid condition [lo]. After 4 hr of incorporation of isotope in uiuo, the DNA of the more dense fraction of nuclei has a higher specific activity than the DNA of the lighter fraction [19]. It has been concluded [lo, 191 that the nuclei of the more dense fraction have increased their content of DNA prior to mitosis, and therefore may be considered to be in the G, phase or in the late part of the S phase of the cell cycle. The less dense nuclei then may be considered to be in the G1 phase or in the early part of the S phase of the cell cycle.
1 This investigation was supported by Public Health Service Research Grant No. CA-01747-13 from the National Cancer Institute and by Grant No. IN-13B from the American Cancer Society. * Predoctoral support provided by the United States Public Health Service Medical Student Research Training Grant MSRT-2R-24 and by Graduate Training Grant 2G-5Tl-GM-157-05 from the National Institutes of Health, United States Public Health Service. The data presented were taken from a thesis submitted to the Faculty of the Graduate School, University of Minnesota, in partial fulfillment of the requirements for the degree of Doctor of Philosophy. Present address: Biochemistry Division, Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Illinois. Experimental
Cell Research 39
436
W. G. Niehaus, Jr. and C. P. Barnum
It has been reported that RNA synthesis is depressed during the period of DNA synthesis (S phase) in Tetrahymena [22], in root tip nuclei [25], in the slime mold [20], and in Euplotes [23], as well as in cultured mammalian cells [24] and in regenerating rat liver nuclei [27]. Nuclear histone, however, appears to be synthesized during the S phase in root tip nuclei [l] and in Euplotes [ll, 231. Experiments have been performed to investigate the metabolism of RNA and histone during the S and G, phases of the cell cycle. The uptake of isotope, in vivo, into RNA and histone has been compared in the two fractions of nuclei, one fraction representing the G, and late part of the S phase, the other representing the G, and the early part of the S phase.
EXPERIMENTAL
PROCEDURE
Male Holtzman rats weighing 250 to 400 g were used in these experiments. Partial hepatectomies were performed by the method of Higgins and Anderson [12] as modified by Brues, Drury and Brues [6]. All partial hepatectomies were performed about 8 a.m. At specified times after partial hepatectomy, 32P-orthophosphate (carrier-free, Nuclear Consultants Corporation) at a level of 1 ,LLC per g of body weight, or 2-W-glycine (Tracerlab) at a level of 0.1 PC per g of body weight, was administered as a single intraperitoneal injection. At various times after administration of isotope the animals were killed and the livers removed. All isolation procedures were performed in a cold room at 4”. The liver was homogenized in a smooth glass homogenizer with a Teflon pestle in 9 volumes of cold 0.25 M sucrose that was 5 mM with respect to CaCl,. In those experiments in which histone was to be isolated, the sucrose solutions were adjusted to pH 5.8 with 0.12 M acetate buffer to minimize proteolysis during the isolation procedure [9]. In the experiments employing snP, a portion of the homogenate was removed for the isolation of acid-soluble nucleotides. Nuclei were isolated from the homogenate by a slight modification of the method of Fisher, Holbrook and Irvin [lo], as described previously [19]. Upon centrifugation through a sucrose density gradient the nuclei were separated into a pellet plus three bands of nuclei. The pellet fraction contains the premitotic nuclei in G, and late S phases, and the lower layer contains the hepatocyte nuclei in G, and early S phases. The middle and upper layers, which contain non-parenchymal nuclei [lo], were not used in these experiments. Preparation
of RNA, acid-soluble
nucleotides, and proteins
In the experiments employing azP, the nucleic acids were extracted from the isolated nuclei and the RNA hydrolyzed and ashed as described previously [19]. Acidsoluble nucleotides were adsorbed onto washed charcoal from the supernatant fluid from the trichloracetic acid precipitation of the homogenate. The charcoal containing the absorbed nucleotides was washed once with distilled water and the nucleotides Experimental
Cell Research 39
Metabolism
of RNA and histone in nuclei prior to mitosis
437
were eluted with a mixture of ethanol, water and concentrated ammonia (50:45:5) and ashed. The specific activities of RNA and acid-soluble nucleotides were determined as described [19]. In those experiments in which histone was to be isolated, the nuclei were suspended in 0.25 N H,SO, and stirred continuously at 4” for 2 hr, then centrifuged 10 min at 600 xg. The pellet, containing nucleic acid and acid-insoluble nuclear protein, was heated with buffered 10 per cent NaCl to solubilize the nucleic acids as described 1191.The nucleic acids were precipitated from the 10 per cent NaCl with two volumes of ethanol and the RNA was removed by alkaline hydrolysis. The DNA was hydrolyzed in 5 per cent trichloroacetic acid and the hydrolysate adsorbed onto charcoal and eluted 1261. Histone was purified by a modification of the method of Laurence, Simson and Butler [15]. The histones were precipitated from the 0.25 N H,SO, by the addition of trichloroacetic acid to a final concentration of 20 per cent. The histone was redissolved in 0.25 N H,SOI by stirring at room temperature for I hr. Some of the protein did not return to solution in this step and was discarded. The histone was again precipitated by addition of trichloroacetic acid to 20 per cent concentration, and the precipitate was redissolved and precipitated. The precipitate was then washed with acetone, dried, and dissolved in 0.004 N HCl. DNA and histone samples were plated directly onto aluminium planchets at infinite thinness, dried and counted on a windowless gas flow counter. DNA was measured by the diphenylamine method of Burton [7] and histone was measured by the method of Lowry, Rosebrougb, Farr and Randall [18], using commercial histone (Worthington) as a standard. RESULTS
Incorporation
of 32P into RNA
When 32P, was administered 18.5 or 27.5 hr after partial hepatectomy (at times near the onset of DNA synthesis and near its peak) and the animals killed 30 min later, the RNA from nuclei in the pellet fraction had a specific activity about 1.5 times that of the RNA from the lower layer of nuclei (Table I). The possibility was considered that the differences in RNA specific activity between the two fractions of nuclei were due to contamination of the nuclei from the lower layer by cytoplasmic RNA, which has a much lower specific activity than does nuclear RNA. Nuclei from the lower layer and from the pellet were isolated and treated with citric acid [3]. As shown in Table II, no significant differences were found between citric acid-treated and untreated nuclei from each fraction. Incorporation
of 14C into DNA and histone
When 2-14C-glycine was administered 26 hr after partial hepatectomy and the animals were killed 2 hr later, the specific activity of histone from the 28 -
651816
Experimental
Cell Research 39
438
W. G. Niehaus, Jr. and C. P. Barnum
pellet fraction of nuclei was 1.6 times that from the lower layer of nuclei, while the ratio for DNA was 2.1. This result with DNA confirmed earlier work using 32Pi as an isotopic precursor and allowed the comparison of DNA and histone in the same animal. When isotope was administered 17 hr TABLE
I. Comparison
of specific activities
of RNA from two fractions
sePi, 1 ,uc per g of body weight, was injected at the indicated later. Each line represents the pooled livers of two rats. Time of isotope administration (hr after partial hepatectomy) 18.5 18.5 18.5 27.5 27.5 27.5 27.5 27.5 27.5
Specific activity (per cent of acid-soluble nucleotide activity) r-h-7 Pellet Lower 26 25 26 18 20 25 17 16 14
37 36 40 23 27 37 27 28 29
time. Animals
of nuclei.
were killed
30 min
Ratio of specific activity Pellet/Lower 1.4 1.4 1.5 1.3 1.3 1.5 1.6 1.7 2.1
after partial hepatectomy and the animals were killed 2 hr later, the specific activity of histone from the pellet fraction was also 1.6 times that from the lower layer of nuclei (Table III).
IHSCUSSION
Incorporation
of 32P into RNA
As shown in Tables I and II, the specific activity of RNA from the pellet fraction of nuclei is greater than that of RNA from the lower nuclear layer, when isotope is administered at two different times after partial hepatectomy. Data in Table II have been interpreted as demonstrating that the smaller RNA specific activity in the lower nuclear layer is not due to contamination by cytoplasmic RNA. Citric acid treatment in the process of isolation of nuclei is known to remove nearly all cytoplasmic contamination, resulting in a progressive increase in the specific activity of the RNA of the nuclear Experimental
Cell Research 39
Metabolism
of RNA and histone in nuclei prior to mitosis
439
preparation [a]. Since no increase in RNA specific activity was seen in these isolated nuclei following subsequent citric acid treatment, it was concluded that the samples of nuclei were not contaminated by cytoplasmic RNA. In order to compare the rates of RNA synthesis at the two time points TABLE
II. Comparison of specific activities of RNA from two fractions before and after citric acid treatment.
asPi, 1 pc per g of bodv weight, was injected 27.5 hr after partial hepatectomy. killed 30 min later. Each line represents the pooled livers of four rats. Specific activity (per cent of acid-soluble nucleotide activity) Lower before citric acid 13 20 20
Lower after citric acid 14 20 17
Pellet before citric acid 17 26
Ratio Pellet after citric acid 16 26 -
Lower after citric acid/ Lower before citric acid 1.1 1.0 0.9
of nuclei
Animals
were
of specific activity Pellet after citric acid/ Pellet before citric acid 0.9 1.0 .-
Pellet/ Lower (untreated nuclei) 1.3 1.3 -
employed, RNA specific activity was calculated as a per cent of the specific activity of acid soluble nucleotide phosphorus, which is presumed to be representative of the precursor pool. The results are consistent with the finding of Welling and Cohen [27] that nuclear RNA synthesis is more rapid 19 hr after partial hepatectomy than after 28 hr. The pellet fraction of nuclei is proposed to consist of two classes of nuclei; those which are in the process of DNA synthesis (S phase), and those which have completed DNA synthesis prior to mitosis (G, phase) [19]. Therefore, the increased RNA specific activity in the pellet fraction over that of the lower layer of nuclei may be due either to a coincident synthesis of RNA and DNA, or to an increase in the rate of RNA synthesis in the period between the end of DNA synthesis and the onset of mitosis (G, phase). As has been mentioned, RNA synthesis is known to be depressed during DNA synthesis in several invertebrate organisms [20, 22, 23, 251. Welling and Cohen [27] have found that in regenerating rat liver, peak synthesis of nuclear RNA occurs about 16 hr after partial hepatectomy and falls off rapidly, reaching a very low level by 24 hr, when DNA synthesis is maximal. Reiter and Littlefield [24], studying cultures of mouse fibroblasts partially synchronized with 5-fluoroExperimental
Cell Research 39
440
W. G. Niehaus, Jr. and C. P. Barnum
deoxyuridine, found the synthesis of nuclear RNA to be depressed somewhat during the period of DNA synthesis, increasing markedly as the latter slowed, and remaining active until the onset of mitosis. Therefore, the more likely interpretation of the data presented in Table I seems to be that the higher TABLE
III.
Comparison
of specific activities of histone fractions of nuclei.
and DNA from
2-‘Gglycine, 0.1 ,uc per g of body weight, was injected at the indicated killed 2 hr later. Each line represents the pooled livers of two rats. Time of isotope administration (hr after partial hepatectomy) 17 17 17 17 26 26 26 26 26 26
time.
Animals
two were
Specific activity DNA (c.p.m./mg DNA) Lower
Pellet
1320 1520 1010 1775
2870 3300 2120 3630
Histone (c.p.m./mg protein) Lower
Pellet
3670 2640 3690 4950 3600 1440 3590 2970 2770 2530
4770 4180 5840 9100 4270 1820 5040 5020 5200
Ratio of specific activity c-A-. DNA Histone Pellet/Lower Pellet/Lower
-
2.2 2.2 2.1 2.0
1.3 1.6 1.6 1.8 1.2 1.3 1.6 1.7 1.8 2.1
RNA specific activity in the pellet fraction of nuclei is due to an increase in nuclear RNA synthesis following the completion of DNA synthesis, i.e., during the G, phase of the cell cycle. Incorporation
of 14C into histone
Reports in the literature indicate that histone and DNA4 are synthesized simultaneously in root tip nuclei [l] and in Euplotes [ll, 231. The time of histone synthesis in mammalian tissues, however, is not clearly defined. Several studies performed using regenerating rat liver have yielded ambiguous results. Holbrook et al. [13] concluded that histone and DNA are synthesized at the same time. Butler and Cohn [S] found the peak of histone synthesis to precede that of DNA. Bloch and Godman [5] originally concluded that histone and DNA are synthesized simultaneously; however, Bloch [4] later reversed this position and stated that a more likely interpretation of his Experimental
Cell Research 39
Metabolism
of RNA and hisfone in nuclei prior
to mitosis
441
earlier work was that histone was synthesized earlier and associated with the DNA immediately after DNA synthesis. There are no indications in the literature that DNA synthesis could precede histone synthesis. Bloch and Godman [5] found newly synthesized DNA complexed with histone even when the nucleus contained an amount of DNA intermediate between diploid and tetraploid. Such an intermediate nucleus is presumed to be in the process of DNA synthesis. Therefore, histone must have been synthesized before or at the time of DNA synthesis. The specific activity of histone was found to be greater in the nuclei of the pellet fraction than in the nuclei of the lower layer after a two-hour period of isotope incorporation in vivo (Table III). It has been proposed that the nuclei in the pellet fraction are in the G, phase or the late part of the S phase of the cell cycle, having increased their content of DNA prior to mitosis. If histone were synthesized earlier than DNA, histone synthesis in these nuclei of the pellet fraction would have been completed before the addition of isotope, and the specific activity of histone in these nuclei would be expected to be low. Since, in fact, the specific activity of histone is found to be higher in these nuclei, it would appear that in the regenerating liver, histone synthesis is increased either during the S phase or the G, phase of the cell cycle. As was mentioned above, it seems unlikely that histone synthesis follows the synthesis of DNA; therefore, the more likely interpretation of the data presented in Table III seems to be that the higher specific activity of histone in the pellet fraction of nuclei is due to an increase in histone synthesis during the period of DNA synthesis, i.e., during the S phase of the cell cycle. Histone as isolated from nuclear fractions by the method of Lawrence et al. [15] would also be expected to contain small amounts of other basic proteins, e.g., those associated with nuclear ribosomes. Therefore the possibility must be considered that the increased incorporation of radioactivity may also reflect an increased synthesis of these basic proteins.
SUMMARY
Following the administration of radioisotope in vivo, nuclei were isolated from regenerating rat liver and separated into two fractions, one of which consisted of nuclei which had increased their DNA content preparatory to mitosis. In this premitotic fraction of nuclei, both RNA and histone were more highly labeled than in the fraction of nuclei with a normal DNA content. Experimental
Cell Research 39
442
W. G. Niehaus, Jr. and C. P. Barnum
It was concluded that histone and DNA are synthesized during the same period, and that there is an increase in the synthesis of nuclear RNA immediately following the cessation of DNA synthesis. REFERENCES 1. ALFERT, M., Exptl Cell Res. Suppl. 6, 227 (1959). C; P.,*Unpublished observations.‘ 2. BARNUM, 3. BARNUM, C. P., NASH. C. W.. JENNINGS, E., NYGAARD, 0. and VERMUND, H., Arch. Bio&m. 25;376 (i950). 4. BLOCH, D. P., J. Cell. Comp. Physiol. 62 Suppl. I, 87 (1963). 5. BLOCH, D. P. and GODMAN, G. C., J. Biophys. Biochem. Cytol. 1, 17 (1955). 6. BRUES, A. M., DRURY, D. R. and BRUES, M. C., Arch. Pathol. 22, 658 (1936). 7. BURTON, K., Biochem. J. 62, 315 (1956). 8. BUTLER, J. A. V. and COHN, P., Biochem. J. 87, 330 (1963). 9. DOUNCE, A. L. and UMANA, R., Biochem. 1, 811 (1962). 10. FISHER, k. F., HOLBROOK, D. J. and IRVIN, J. L:, J. Cell. Biol. 17, 231 (1963). 11. GALL, J. G., J. Biophys. Biochem. Cytol. 5, 295 (1959). G. M. and ANDERSON, R. M., Arch. Pathol. 12, 186 (1931). 12. HIGGINS, D. J., EVANS, J. H. and IRVIN, J. L.. Erptl Cell Res. 28, 120 (1962). 13. HOLBROOK, 14. HOWARD, A. and’PE~c, S: R., Heredity SuppI. 6, ‘261’(1953). D. J. R., SIMSON, P. and BUTLER, J. A. V., Biochem. .7. 87, 200 (1963). 15. LAURENCE, 16. LOONEY, W. B., Proc. Nat1 Acad. Sci. U.S. 46, 690 (1960). in J. S. MITCHELL (ed.), The Cell Nucleus, p. 98. Academic Press, New York, 1960. 17. ~ N. J., FARR, A. L. and RANDALL, R. J., J. Biol. Chem. 193, 18. LOWRY, 0. H., ROSEBROUGH, 265 (1951). W. G. and BARNUM, C. P., J. Biot. Chem. 239, 1198 (1964). 19. NIEHAUS, 20. NYGAARD, 0. F., G~~TTES, S. and RUSCH, H. P., Biochim. Biophys. Acta 38, 298 (1960). 21. PRESCOTT, D. M., Intern. Reu. Cytol. 11, 255 (1961). 22. __ Exptl Cell Res. 19, 228 (1960). R. F.. Proc. Natl Acad. Sci. U.S. 47. 686 (1961). 23. PRESCOTT. D. M. and KIMBALL, 24. REITER, j. M. and LITTLEFIELD, J. w., Biochim. Biophys. Acta 80, 562 (1664).’ 25. SISKEN, J. E., Expfl Cell Res. 16, 602 (1959). 26. Snnm, I. (ed.), Chromatographic and Electrophoretic Techniques, Vol. 1, p. 242. Intersciencc Publishers, New York, 1960. 27. WELLING, W. and COHEN, J. A., Biochim. Biophys. Acta 42, 181 (1960).
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