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Additive effect of histones and actinomycin D on the cellular RNA synthesis
454
Experimental
Cell Research 42, 484-189 (1966)
ADDITIVE EFFECT OF HISTONES AND ACTINOMYCIN D ON THE CELLULAR RNA SYNTHESIS A. G. BUKRINSKAYA, 0. BURDUCEA, and T. A. ASSADULLAEV The Institute
of Virology,
USSR Academy of Medical
A. K. GITELMAN Sciences, Moscow,
USSR
Received November 9. 1965
HISTONESare
known to inhibit the cellular RNA synthesis both in z&o and [ 1,2,7]. This effect appears to be due to the complex formation between histone and DNA and to the blockage of DNA template function in RNA synthesis by RNA polymerase [2, 71. Actinomycin D was also shown to bind with DNA and to inhibit enzymatic DNA-dependent RNA synthesis [3, lo]. The present study was undertaken, first, to determine how homologous and heterologous histones affect RNA synthesis in tissue culture cells, and, secondly, to estimate the relationship between binding sites on DNA for histones and actinomycin D. in vitro
MATERIALS
AND
METHODS
Cells
Primary chick embryo cultures prepared from II-day-old eviscerated embryos and containing 5 x 10s cells per culture and HEp-2 cell cultures containing I x 10s cells per culture were used as monolayers to assay the rate of RNA synthesis. HEp-2 cells grown on the strips of slides and used when the monolayer had not been fully formed were employed for autoradiographic studies. Histones
The total histone from calf thymus gland was isolated according to the method of Hnilica and Hupca [6] and kindly supplied by Dr V. Shpikiter. F2a histone fraction was isolated according to the method of Johns and Butler [S] and kindly supplied by Dr V. Vorobyev. The total chick embryo histone was isolated from the nuclei of trypsinized cells of 13-15-day-old eviscerated chick embryos with 0.25 N HCl after preliminary extraction of globulins with 0.14 M NaCl solution. The histone was precipitated by 3 volumes of acetone, washed twice with acetone and dried by lyophylization. For Experimental
Cell Research 42
Effect of histones and actinomycin
D on RNA synthesis
485
labeling histone the cells were incubated in buffer sait solution (BSS) with 2.5 &/ml of %-leucine (specific activity 0.5 mC/mg) for 1 hr prior to protein extraction. All preparations of histones were used at the concentration of loo-150 pg/ml which did not provoke cell destruction after 3 hr incubation. Actinomycin D was given as a gift by Merck, Sharpe and Dohme Co., USA, and used at the concentration of 2.0 pg/ml. Assay of histone and actinomycin D effect on RNA
synthesis
Samples of cell monolayers were overlaid with BSS containing actinomycin D (2 pg/ml) or histone (loo-150 pg/ml). In some experiments histone was added 30 min before actinomycin or actinomycin was added 30 min before histone. The controls were treated with actinomycin or with histone added at appropriate times. WZ-Cytidine (specific activity 2.44 mC/mmole) was added to the medium at the concentration of 0.5 or 1.0 &/ml after 30 min of incubation for 1 or 1.5 hr period of incubation. Then the medium was removed, cells washed with BSS, taken off the glass, treated on millipore membranes with cold 4 per cent HClO, and alcohol and dried. Counts of radioactivity were carried out with a counter of Geiger-Miiller type. In some cases membranes after counting were dissolved in acetone, and RNA was hydrolysed by 1 M KOH for 18 hr at 37°C. Measurements of RNA were made according to the method of Spirin [11], and the radioactivity was recounted per 1 pg of RNA. In other cases the average radio activity per one monolayer was calculated. Assay of labeled histone penetration
into cells
HEp-2 cells were exposed to 500 yg/ml of chick embryo histone labeled with laCleucine (specific activity of histone 566 cpm/mg). To the control cells l*C-leucine (specific activity 0.5 mC/mg) was added at such concentration that the radioactivity of solution was similar to that of histone solution. After 15, 30 and 60 min of incubation the samples of cells were washed, fixed with Carnoy solution, treated with cold 5 per cent TCA for 1 hr at +4X and stored at +4X for autoradiographic exposure. After 8 days the slides were developed, washed and dried. The developed specimens were stained with methylene blue. RESULTS Penetration
of labeled histone into the cell
The autoradiographic experiments were performed in order to determine the rate of labeled histone penetration into HEp-2 cells. When the contact with labeled histone lasted for 15 min, the developed silver grains were seen over the nuclei concentrating around the nucleoli, while only few grains were found over the cytoplasm (Table I, Fig. 1). A considerable number of cells contained the grains only around the nucleoli (Fig. 1 c). When contact with labeled histone lasted for 30 min, the number of grains was the same as Experimental
Cell Research 42
486
A. G. Bukrinskaya,
0. Burducea, A. Ii. Gitelman
und T. A. Assadullaeu
Fig. I.-Autoradiographs of HEp-2 cells exposed for 15 min to A I%-leucine (specific activity 500 $/mg), B and C ‘*C-histone (specific activity 566 cpm/mg). x 600.
for the 15 min of exposure. Only few labeled cells were detected after a 1 hr period of contact with labeled histone. When the cells were exposed to 14C-leucine most of the grains were seen over the cytoplasm and only few grains were found over the nucleoli (Table I, Fig. 1). Effect of histones and actinomycin
II on RNA synthesis
From Fig. 2 it may be seen that at the concentration of 2 ,ug/ml actinomycin D suppressed the 14C-cytidine incorporation into chick fibroblasts by more than 90 per cent, the inhibition being maximal after 2-3 hr of incubation. The same degree of inhibition of RNA synthesis was observed in actinomycin-treated HEp-2 cells. TABLE
I. The incorporation
of 14C-leucine and histone labeled with 14C-leucine into HEp-2 cells. Number of grains
W-leucine WZ-histone
Cytoplasm
Karyoplasm
Nucleolus
39.1+ 2.7 3.8 i: 0.63
19.1 f 1.8 4.110.5
4.1 I!z0.7 10.1 kO.4
500 pg/ml of “C-leucine labeled chick embryo histone (specific activity 566 cpm/mg) and I%leucine (specific activity 0.5 mC/mg) at the doses with the same radioactivity as histone were added to HEp-2 cells. After 15 min of incubation the cells were washed and fixed. Autoradiographs were prepared as described under Methods. Experimental
Cell Research 42
Effect of histones and actinomycin
D on RNA synthesis
487
The total calf thymus histone suppressed RNA synthesis in chick tibroblasts less than actinomycin D (Fig. 2). The f2a histone fraction produced a stronger inhibitory effect than did the total histone (Fig. 3). The histone treated with 0.5 per cent trypsin solution did not exhibit inhibitory effect (Fig. 3). The total chick embryo histone produced the same inhibitory effect on RNA synthesis in chick Iibroblasts as the calf thymus histone. Both kinds of histones inhibited the RNA synthesis to a more pronounced effect in the chick fibroblasts than in HEp-2 cells (Fig. 4 and Table II). Thus, we did not reveal any specificity in homologous histone action on RNA synthesis. Combined effect of actinomycin D and histones When tissue cultures were treated both with actinomycin D and with histones the inhibition of RNA synthesis was stronger than that produced by
--
\ 30-
1 \
20-
‘, f '.
lo-
‘+*-I
0
12
3
Fig. 2.
4hr
0
1
2
3hr
o-0
Fig. 3.
12
3hr
Fig. 4.
Fig. 2.-Effect of actinomycin D (*- --*) and calf thymus histone (e-.) on RNA synthesis in chick fibroblasts. The cultures were kept at + 4°C for 1 hr, then 2 pg/ml of actinomycin D and 100 or 150 pg/ml of histone were added to the cells in BSS, and the cultures were transferred to 37”. 0.5 &/ml of W-cytidine (specific activity 2.44 mC/mmole) was added at hourly intervals for 1 hr and the radioactivity was determined in acid-insoluble fraction. Fig. 3.-Effect of cell thymus on RNA synthesis in chick fibroblasts. 150 pg/ml of histone in BSS was added to the cells prior to incubation. For trypsin treatment 1500 pg/ml of histone was mixed with equal volume of 0.5 y0 trypsin Difco and incubated at 37” for 1 hr. Then tenfold diluted. 0.5 &/ml of W-cytidine was added to the cells, and the cultures were incubated for 2 hr, then the radioactivity of acid-insoluble fraction was determined. a--* total histone; * - * - A f 2a fraction; a--* trypsin treated histone. Fig. 4. Effect of f2a histone fraction from calf thymus on RNA synthesis in chick fibroblasts (.-*) and HEp-2 cells (0 - - - *). For explanations see Fig. 2. Experimental
Cell Research 42
488
A. G. Bukrinskaya,
0. Burducea, A. K. Gitelman and T. A. Assaduilaev
sole actinomycin D and by sole histone (Table 11~). The same situation was observed when the cells were first treated with histones and then with actinomycin D (Table IIb). TABLE
II. Effect of acfinomycin D and histones on l*C-cyfidine info tissue culture cells. HEp-2
Chick fibroblasts
incorporation cells
Exp. 1 Exp. 2
wm/Pg RNA
%
cpmlyg RNA
%
wm
%
1804 102 570 34
100 5.2 31.5 -
PART A
Control Actinomycin Calf thymus f2a in 30 min Chick embryo histone in 30 min Actinomycin, then f2a Actinomycin, then chick embryo histone
612 62 60 195 22
100 10.1 9.8 31.7 3.5
213 45 117 158 20
100 21.1 55.0 74.1 9.3
33
5.4
37
17.6
-
1.8
-
Chick fibroblasts hours after incubation of contact with W-cytidine 0.5-1.5 hr 1.5-2.5 hr 2.5-3.5 hr PART B
cm
Control Calf thymus f2a Actinomycin in 30 min f2a, then actinomycin
844 292 211 9
%
100 34.6 25.0 1.0
wm
1014 257 84 18
%
100 25.3 8.2 1.7
wm
%
853 230 40 32
100 26.9 4.6 3.7
Part A. Histone (150 pg/ml): 30 min before actinomycin D (2 pg/ml). I%-cytidine (1.0 ,&/ml) was added simultaneously with histone for 1.5 hr, then radioactivity of acid-insoluble fraction was determined. Part B. Actinomycin D (2 pg/ml): 30 min before histone (150 ,ug/ml). W-cytidine (0.5 &/ml) was added in 0.5 hr, 1.5 hr, and 2.5 hr of incubation for 1 hr period, then the radioactivity of acid-insoluble fraction was determined. DISCUSSION
The data presented show that exogenic histone penetrates into the cell concentrating around nucleoli. Less label was found over karyoplasm and cytoplasm; it is not known whether this label should be attributed to penetrated histone or to histone which had been adsorbed on cell surface. It is noteworthy that actinomycin D also binds at first with nucleolar DNA which contains more guanine than DNA of nuclear chromatin [4, 91. The total chick embryo and calf thymus histones equally suppressed Experimental
Cell Research 42
Effect of histones an actinomycin
D on RNA synthesis
489
synthesis in chick fibroblasts, the inhibitory effect of both kinds of histones being higher than in HEp-2 cells. Thus we did not reveal any specificity in homologous histone action, this finding being in accordance with the suggestion that interaction between histone and DNA is nonspecific [l]. The differences in histone action on RNA synthesis in the two kinds of tissue culture cells appear to be due to the peculiarities of their metabolism. It is difficult to interpret the additive effect of actinomycin D and histone on RNA synthesis since the mode of histone attachment to DNA has not yet been revealed. If it is assumed that exogenic histone binds with DNA, it is likely that histone-binding sites involve bases in DNA other than guanine, since complex formation between actinomycin D and DNA shows an absolute specificity requirement for this base [3]. According to X-ray studies, actinomycin D is visualized as bound in the minor groove of helical DNA with which it forms up to seven hydrogen bonds [S]. Most of the histone is bound with DNA as an interrupted wound around the major groove of DNA forming a bridged network [12]. It may be suggested that the additive effect of actinomycin D and histone on RNA synthesis is based on stereochemical differences of their binding with DNA. SUMMARY
Chick embryo histone labeled with 14C-leucine was shown to penetrate into the cell and to concentrate around nucleoli. Both kinds of histone, isolated from chick embryos and from calf thymus, equally suppressed the RNA synthesis in chick fibroblasts. Less inhibition of RNA synthesis was observed in HEp-2 cells. It was shown that actinomycin and histone produced the additive inhibitory effect on RNA synthesis in tissue culture cells. REFERENCES 1. ALLFREY, V. G. and MIRSKY, A. E., in Bonner and Ts’o (eds), Nucleohistones, Holden Day, New York, 1964. 2. BARR, G. C. and BUTLER, J. A., Nature 199, 1170 (1963). 3. GOLDBERG, I. H., RABINOWITZ, M. and REICH, E., Proc. Nat1 Acad. Sci. U.S.A. 48,2094 (1962). 4. GRANBOULAN, N. and GRANBOULAN, P., Exptl Cell Res. 38, 604 (1965). 5. HAMILTON, L., FULLER, W. and REICH, E., Nature 198, 538 (1963). 6. HNILICA, 2. and HUPKA, S., Biologia (Czechoslovakia) 11, 821 (1959). 7. HUANG, R. and BONNER, J., Proc. Natl. Acad. Sci. U.S.A. 48, 1216 (1962). 8. JOHNS, W. E. and BUTLER, J. A., Biochem. J. 84, 436 (1962). 9. MAGGIO, R., SIEKEVITZ, P. and PALADE, G., J. Cell. Biol. 18, 293 (1963). 10. REICH, E., FRANCLIN, R. M., SHATKIN, A. J. and TATUM, E. L., Science 134, 556 (1961). 11. SPIRIN, A. N., Biochem. 23, 656 (1958). In Russian. 12. ZUBAY, G. and DOTY, P., J. Mol. Biol. 1, 1 (1959).
Experimental
Cell Research
42
Experimental
Cell Research 42, 484-189 (1966)
ADDITIVE EFFECT OF HISTONES AND ACTINOMYCIN D ON THE CELLULAR RNA SYNTHESIS A. G. BUKRINSKAYA, 0. BURDUCEA, and T. A. ASSADULLAEV The Institute
of Virology,
USSR Academy of Medical
A. K. GITELMAN Sciences, Moscow,
USSR
Received November 9. 1965
HISTONESare
known to inhibit the cellular RNA synthesis both in z&o and [ 1,2,7]. This effect appears to be due to the complex formation between histone and DNA and to the blockage of DNA template function in RNA synthesis by RNA polymerase [2, 71. Actinomycin D was also shown to bind with DNA and to inhibit enzymatic DNA-dependent RNA synthesis [3, lo]. The present study was undertaken, first, to determine how homologous and heterologous histones affect RNA synthesis in tissue culture cells, and, secondly, to estimate the relationship between binding sites on DNA for histones and actinomycin D. in vitro
MATERIALS
AND
METHODS
Cells
Primary chick embryo cultures prepared from II-day-old eviscerated embryos and containing 5 x 10s cells per culture and HEp-2 cell cultures containing I x 10s cells per culture were used as monolayers to assay the rate of RNA synthesis. HEp-2 cells grown on the strips of slides and used when the monolayer had not been fully formed were employed for autoradiographic studies. Histones
The total histone from calf thymus gland was isolated according to the method of Hnilica and Hupca [6] and kindly supplied by Dr V. Shpikiter. F2a histone fraction was isolated according to the method of Johns and Butler [S] and kindly supplied by Dr V. Vorobyev. The total chick embryo histone was isolated from the nuclei of trypsinized cells of 13-15-day-old eviscerated chick embryos with 0.25 N HCl after preliminary extraction of globulins with 0.14 M NaCl solution. The histone was precipitated by 3 volumes of acetone, washed twice with acetone and dried by lyophylization. For Experimental
Cell Research 42
Effect of histones and actinomycin
D on RNA synthesis
485
labeling histone the cells were incubated in buffer sait solution (BSS) with 2.5 &/ml of %-leucine (specific activity 0.5 mC/mg) for 1 hr prior to protein extraction. All preparations of histones were used at the concentration of loo-150 pg/ml which did not provoke cell destruction after 3 hr incubation. Actinomycin D was given as a gift by Merck, Sharpe and Dohme Co., USA, and used at the concentration of 2.0 pg/ml. Assay of histone and actinomycin D effect on RNA
synthesis
Samples of cell monolayers were overlaid with BSS containing actinomycin D (2 pg/ml) or histone (loo-150 pg/ml). In some experiments histone was added 30 min before actinomycin or actinomycin was added 30 min before histone. The controls were treated with actinomycin or with histone added at appropriate times. WZ-Cytidine (specific activity 2.44 mC/mmole) was added to the medium at the concentration of 0.5 or 1.0 &/ml after 30 min of incubation for 1 or 1.5 hr period of incubation. Then the medium was removed, cells washed with BSS, taken off the glass, treated on millipore membranes with cold 4 per cent HClO, and alcohol and dried. Counts of radioactivity were carried out with a counter of Geiger-Miiller type. In some cases membranes after counting were dissolved in acetone, and RNA was hydrolysed by 1 M KOH for 18 hr at 37°C. Measurements of RNA were made according to the method of Spirin [11], and the radioactivity was recounted per 1 pg of RNA. In other cases the average radio activity per one monolayer was calculated. Assay of labeled histone penetration
into cells
HEp-2 cells were exposed to 500 yg/ml of chick embryo histone labeled with laCleucine (specific activity of histone 566 cpm/mg). To the control cells l*C-leucine (specific activity 0.5 mC/mg) was added at such concentration that the radioactivity of solution was similar to that of histone solution. After 15, 30 and 60 min of incubation the samples of cells were washed, fixed with Carnoy solution, treated with cold 5 per cent TCA for 1 hr at +4X and stored at +4X for autoradiographic exposure. After 8 days the slides were developed, washed and dried. The developed specimens were stained with methylene blue. RESULTS Penetration
of labeled histone into the cell
The autoradiographic experiments were performed in order to determine the rate of labeled histone penetration into HEp-2 cells. When the contact with labeled histone lasted for 15 min, the developed silver grains were seen over the nuclei concentrating around the nucleoli, while only few grains were found over the cytoplasm (Table I, Fig. 1). A considerable number of cells contained the grains only around the nucleoli (Fig. 1 c). When contact with labeled histone lasted for 30 min, the number of grains was the same as Experimental
Cell Research 42
486
A. G. Bukrinskaya,
0. Burducea, A. Ii. Gitelman
und T. A. Assadullaeu
Fig. I.-Autoradiographs of HEp-2 cells exposed for 15 min to A I%-leucine (specific activity 500 $/mg), B and C ‘*C-histone (specific activity 566 cpm/mg). x 600.
for the 15 min of exposure. Only few labeled cells were detected after a 1 hr period of contact with labeled histone. When the cells were exposed to 14C-leucine most of the grains were seen over the cytoplasm and only few grains were found over the nucleoli (Table I, Fig. 1). Effect of histones and actinomycin
II on RNA synthesis
From Fig. 2 it may be seen that at the concentration of 2 ,ug/ml actinomycin D suppressed the 14C-cytidine incorporation into chick fibroblasts by more than 90 per cent, the inhibition being maximal after 2-3 hr of incubation. The same degree of inhibition of RNA synthesis was observed in actinomycin-treated HEp-2 cells. TABLE
I. The incorporation
of 14C-leucine and histone labeled with 14C-leucine into HEp-2 cells. Number of grains
W-leucine WZ-histone
Cytoplasm
Karyoplasm
Nucleolus
39.1+ 2.7 3.8 i: 0.63
19.1 f 1.8 4.110.5
4.1 I!z0.7 10.1 kO.4
500 pg/ml of “C-leucine labeled chick embryo histone (specific activity 566 cpm/mg) and I%leucine (specific activity 0.5 mC/mg) at the doses with the same radioactivity as histone were added to HEp-2 cells. After 15 min of incubation the cells were washed and fixed. Autoradiographs were prepared as described under Methods. Experimental
Cell Research 42
Effect of histones and actinomycin
D on RNA synthesis
487
The total calf thymus histone suppressed RNA synthesis in chick tibroblasts less than actinomycin D (Fig. 2). The f2a histone fraction produced a stronger inhibitory effect than did the total histone (Fig. 3). The histone treated with 0.5 per cent trypsin solution did not exhibit inhibitory effect (Fig. 3). The total chick embryo histone produced the same inhibitory effect on RNA synthesis in chick Iibroblasts as the calf thymus histone. Both kinds of histones inhibited the RNA synthesis to a more pronounced effect in the chick fibroblasts than in HEp-2 cells (Fig. 4 and Table II). Thus, we did not reveal any specificity in homologous histone action on RNA synthesis. Combined effect of actinomycin D and histones When tissue cultures were treated both with actinomycin D and with histones the inhibition of RNA synthesis was stronger than that produced by
--
\ 30-
1 \
20-
‘, f '.
lo-
‘+*-I
0
12
3
Fig. 2.
4hr
0
1
2
3hr
o-0
Fig. 3.
12
3hr
Fig. 4.
Fig. 2.-Effect of actinomycin D (*- --*) and calf thymus histone (e-.) on RNA synthesis in chick fibroblasts. The cultures were kept at + 4°C for 1 hr, then 2 pg/ml of actinomycin D and 100 or 150 pg/ml of histone were added to the cells in BSS, and the cultures were transferred to 37”. 0.5 &/ml of W-cytidine (specific activity 2.44 mC/mmole) was added at hourly intervals for 1 hr and the radioactivity was determined in acid-insoluble fraction. Fig. 3.-Effect of cell thymus on RNA synthesis in chick fibroblasts. 150 pg/ml of histone in BSS was added to the cells prior to incubation. For trypsin treatment 1500 pg/ml of histone was mixed with equal volume of 0.5 y0 trypsin Difco and incubated at 37” for 1 hr. Then tenfold diluted. 0.5 &/ml of W-cytidine was added to the cells, and the cultures were incubated for 2 hr, then the radioactivity of acid-insoluble fraction was determined. a--* total histone; * - * - A f 2a fraction; a--* trypsin treated histone. Fig. 4. Effect of f2a histone fraction from calf thymus on RNA synthesis in chick fibroblasts (.-*) and HEp-2 cells (0 - - - *). For explanations see Fig. 2. Experimental
Cell Research 42
488
A. G. Bukrinskaya,
0. Burducea, A. K. Gitelman and T. A. Assaduilaev
sole actinomycin D and by sole histone (Table 11~). The same situation was observed when the cells were first treated with histones and then with actinomycin D (Table IIb). TABLE
II. Effect of acfinomycin D and histones on l*C-cyfidine info tissue culture cells. HEp-2
Chick fibroblasts
incorporation cells
Exp. 1 Exp. 2
wm/Pg RNA
%
cpmlyg RNA
%
wm
%
1804 102 570 34
100 5.2 31.5 -
PART A
Control Actinomycin Calf thymus f2a in 30 min Chick embryo histone in 30 min Actinomycin, then f2a Actinomycin, then chick embryo histone
612 62 60 195 22
100 10.1 9.8 31.7 3.5
213 45 117 158 20
100 21.1 55.0 74.1 9.3
33
5.4
37
17.6
-
1.8
-
Chick fibroblasts hours after incubation of contact with W-cytidine 0.5-1.5 hr 1.5-2.5 hr 2.5-3.5 hr PART B
cm
Control Calf thymus f2a Actinomycin in 30 min f2a, then actinomycin
844 292 211 9
%
100 34.6 25.0 1.0
wm
1014 257 84 18
%
100 25.3 8.2 1.7
wm
%
853 230 40 32
100 26.9 4.6 3.7
Part A. Histone (150 pg/ml): 30 min before actinomycin D (2 pg/ml). I%-cytidine (1.0 ,&/ml) was added simultaneously with histone for 1.5 hr, then radioactivity of acid-insoluble fraction was determined. Part B. Actinomycin D (2 pg/ml): 30 min before histone (150 ,ug/ml). W-cytidine (0.5 &/ml) was added in 0.5 hr, 1.5 hr, and 2.5 hr of incubation for 1 hr period, then the radioactivity of acid-insoluble fraction was determined. DISCUSSION
The data presented show that exogenic histone penetrates into the cell concentrating around nucleoli. Less label was found over karyoplasm and cytoplasm; it is not known whether this label should be attributed to penetrated histone or to histone which had been adsorbed on cell surface. It is noteworthy that actinomycin D also binds at first with nucleolar DNA which contains more guanine than DNA of nuclear chromatin [4, 91. The total chick embryo and calf thymus histones equally suppressed Experimental
Cell Research 42
Effect of histones an actinomycin
D on RNA synthesis
489
synthesis in chick fibroblasts, the inhibitory effect of both kinds of histones being higher than in HEp-2 cells. Thus we did not reveal any specificity in homologous histone action, this finding being in accordance with the suggestion that interaction between histone and DNA is nonspecific [l]. The differences in histone action on RNA synthesis in the two kinds of tissue culture cells appear to be due to the peculiarities of their metabolism. It is difficult to interpret the additive effect of actinomycin D and histone on RNA synthesis since the mode of histone attachment to DNA has not yet been revealed. If it is assumed that exogenic histone binds with DNA, it is likely that histone-binding sites involve bases in DNA other than guanine, since complex formation between actinomycin D and DNA shows an absolute specificity requirement for this base [3]. According to X-ray studies, actinomycin D is visualized as bound in the minor groove of helical DNA with which it forms up to seven hydrogen bonds [S]. Most of the histone is bound with DNA as an interrupted wound around the major groove of DNA forming a bridged network [12]. It may be suggested that the additive effect of actinomycin D and histone on RNA synthesis is based on stereochemical differences of their binding with DNA. SUMMARY
Chick embryo histone labeled with 14C-leucine was shown to penetrate into the cell and to concentrate around nucleoli. Both kinds of histone, isolated from chick embryos and from calf thymus, equally suppressed the RNA synthesis in chick fibroblasts. Less inhibition of RNA synthesis was observed in HEp-2 cells. It was shown that actinomycin and histone produced the additive inhibitory effect on RNA synthesis in tissue culture cells. REFERENCES 1. ALLFREY, V. G. and MIRSKY, A. E., in Bonner and Ts’o (eds), Nucleohistones, Holden Day, New York, 1964. 2. BARR, G. C. and BUTLER, J. A., Nature 199, 1170 (1963). 3. GOLDBERG, I. H., RABINOWITZ, M. and REICH, E., Proc. Nat1 Acad. Sci. U.S.A. 48,2094 (1962). 4. GRANBOULAN, N. and GRANBOULAN, P., Exptl Cell Res. 38, 604 (1965). 5. HAMILTON, L., FULLER, W. and REICH, E., Nature 198, 538 (1963). 6. HNILICA, 2. and HUPKA, S., Biologia (Czechoslovakia) 11, 821 (1959). 7. HUANG, R. and BONNER, J., Proc. Natl. Acad. Sci. U.S.A. 48, 1216 (1962). 8. JOHNS, W. E. and BUTLER, J. A., Biochem. J. 84, 436 (1962). 9. MAGGIO, R., SIEKEVITZ, P. and PALADE, G., J. Cell. Biol. 18, 293 (1963). 10. REICH, E., FRANCLIN, R. M., SHATKIN, A. J. and TATUM, E. L., Science 134, 556 (1961). 11. SPIRIN, A. N., Biochem. 23, 656 (1958). In Russian. 12. ZUBAY, G. and DOTY, P., J. Mol. Biol. 1, 1 (1959).
Experimental
Cell Research
42