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Studies on in vitro RNA synthesis in rat brain nuclei: biphasic stimulation of RNA polymerase with ammonium sulfate
516
SHORT COMMUNICATIONS
Studies on in vitro RNA syn~sis in rat brain nuclei: biphasic stimulation of RNA polymerase with ammonium sulfate Stimulation of RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) activity by ammonium sulfate has been observed with the 'aggregate' enzyme prepared from a variety of mammalian cell nuclei '~,s. This effect has been suggested to result from (a) unmasking of a latent R N A potymerase activity al, (b) dissociation of histones from the DNA template thus providing more D N A loci for transcription 1, or (c) inhibition of degradative enzymes 1°. A soluble DNA-dependent RNA polymerase activity in rat liver nuclei having metal and pH requirements similar to those of the purified bacterial RNA-polymerase has been reported 9. Contrary to the stimulatory effect of ammonium sulfate upon the 'aggregate' RNA polymerase, the soluble enzyme activity is strongly inhibited when ammonium sulfate is present in the incubation medium prior to enzyme addition 9. This communication describes a biphasic effect of ammonium sulfate on the RNA potymerase activity in rat brain nuclei. Preliminary studies on the enzyme activity in enriched neuronal and glial nuclei are also reported. Albino rats of Sprague-Dawley strain weighing about 200 g were used. The preparation of total brain nuclei was carried out as previously described ;~ except that the cerebellum was removed prior to homogenization. The fractionation of brain nuclei into 'large' (neuronal) and 'small' (glial) nuclei by sucrose density gradient centrifugation was performed by a modification of the method of Lovtrup and McEwenL A discontinuous gradient of 2.2, 2.4 and 2.6 M sucrose solutions was prepared and allowed to stand for I h in the cold. The suspension of brain nuclei in 2.0 M sucrose solution was layered over the gradient and centrifuged in a Spinco SW 25 rotor at 53,000 x g for 40 rain. Phase contrast examination of the band obtained at the interphase of 2.2 and 2.4 M sucrose showed a fairly uniform population of large, oval or round nuclei with one distinct nucleolus. The interphase between 2.4 and 2.6 M sucrose consisted chiefly of small nuclei with dense chromatin. These two nuclear populations were referred to as 'enriched' neuronal and glial nuclei, respectively. The conditions of the enzyme assay and the measurement of radioactivity were as described previously 3 except that the incubations were carried out for 10 rain. D N A was measured by the fluorometric method of Kissane and RobbinsL The effects of ammonium sulfate on RNA polymerase activity in whole brain nuclei in the presence of the optimum amount of MnCI2 (4 raM) are shown in Fig. 1. When the salt was added to the incubation mixture before initiation of R N A synthesis (lower curve) a slight stimulation was observed at low ammonium sulfate concentration and above 0.41 M the enzyme activity was inhibited. However, when ammonium sulfate was added to the complete incubation medium after RNA synthesis had already started (upper curve) a several-fold stimulation of the reaction was observed at an optimal salt concentration of 0.33 M. The rate decreased at higher ammonium sulfate concentration but even at 0.66 M a two-fold stimulation over the control persisted, l f t h e reaction were started by addition of enzyme to the complete incubation mixture containing ammonium sulfate, the stimulation of polymerase Brain Research,
16 (1969) 516-519
SHORT COMMUNICATIONS
5 17
1.5 /o,
/ ,, l o
,
\
J
........
;;S
0.50
0['
0.1
........~ . . . ....
0.2
0.5
ON
0.5
",~
0.6
0.7
0.8
M (NH4) z S04
Fig. I. The incorporation of [aH]CTP by the aggregate R N A polymerase from rat brain nuclei as a function of (NH4)eSO4 concentration. The incubation medium contained (in ffmoles) Tris-HCl, pH 8.1, 40; MnCI~, 1; KCI, 15; NaF, 5; ATP, GTP and UTP, 0.25 each; [aH]CTP (sp. activity, 40 ffC/ffmole) 0.125 and enzyme in a final volume of 0.25 ml. Incubation at 37 ° for 10 min. Addition of (NH4)2804, • - • before initiation of RNA synthesis and C- - -(3 after initiation. × . . . x, Reaction started by addition of the enzyme to complete incubation medium including (NH4)2504. 0.75
E
/
0.50-
o
g
<
x, 0.25 -
o=
o
oi~
d2
ds
o)
o.'s
o:~
o}
M (NH4) 2 S04
Fig. 2. The incorporation of [aH]CTP by the aggregate R N A polymerase from rat brain and liver nuclei as a function of (NHa)2SO4 concentration. (NH4)zSO4 was added after initiation of R N A synthesis. For incubation conditions see legend to Fig. 1. 2. - f,, Brain nuclear enzyme and • •, liver nuclear enzyme.
a c t i v i t y was i n t e r m e d i a t e (Fig. I, m i d d l e curve). In this case also, no i n h i b i t i o n o f a c t i v i t y c o u l d be d e m o n s t r a t e d 1.0 M ( n o t shown).
with a m m o n i u m
sulfate c o n c e n t r a t i o n s as high as
T h e effects o f a m m o n i u m sulfate u p o n the ' a g g r e g a t e ' R N A p o l y m e r a s e a c t i v i t y in brain nuclei are q u a l i t a t i v e l y s i m i l a r to t h o s e o b s e r v e d with purified b a c t e r i a l R N A p o l y m e r a s e 4. M o r e o v e r , u n l i k e liver nuclei '~ the s t i m u l a t i o n o f R N A p o l y m e r a s e in brain nuclei was c o n s i d e r a b l y h i g h e r w h e n the salt was a d d e d a f t e r i n i t i a t i o n o f R N A synthesis t h a n w h e n a m m o n i u m
sulfate was p r e s e n t p r i o r to i n i t i a t i o n . Brain Research, 16 (1969) 516-519
518
SHORT COMMUNICATIONS
TABLE I OF [3H]UTP BY T H E (/,#moles UMP/mg DNA/IO rain)
INCORPORATION
AGGREGATE
RNA
P O L Y M E R A S E F R O M R A T B R A I N N U C I El
Incubation conditions were as described in legend to Fig. 1, except that [3H]CTP was replaced by [sH]UTP.
Complete incubation medium Omit ATP, GTP and CTP Complete ÷ (NH4)~SO4
Unfractionated nuclei
Neuronal nuclei
Glial mtclel
215.2 101.0 1505.4
227.5 88.3 2270.0
160.2 97.7 96 I. 2
Further studies with a finer a m m o n i u m sulfate concentration gradient revealed a biphasic stimulation of R N A polymerase activity in brain nuclei. The results of these studies are shown in Fig. 2. The upper curve depicts the response of the brain nuclear enzyme activity to the addition of a m m o n i u m sulfate after initiation of R N A synthesis. When liver nuclei were incubated under identical conditions a broad peak of stimulation was observed (Fig. 2, lower curve). Regardless of the mechanism of stimulation, brain and liver nuclear RN A polymerase activities respond differentially to a m m o n i u m sulfate under identical conditions of incubation. Under somewhat different incubation conditions than those used in the present investigation, a biphasic stimulation of the liver enzyme activity by a m m o n i u m sulfate has recently been reported 6. Preliminary results of the R N A polymerase activity in brain nuclei (unfractionated, neuronal and glial) are summarized in Table I. A m m o n i u m sulfate was found to increase the incorporation of labeled U T P by all 3 fractions, although the effect appeared to be most marked in the neuronal nuclei. In addition, the incorporation of [zH]UTP in the absence of ATP, CTP and G T P was considerably higher in glial nuclei (61 ~o of that found in the presence of the other 3 nucleoside triphosphates) as compared with neuronal nuclei (39 ~o). Whether the incorporation of labeled U T P in the absence of ATP, G T P and CTP is due to polyuridylate synthesis is uncertain at present. However, under similar conditions of incubation we have observed a high rate of poly U synthesis in human brain tumors of glial origin 3. The authors are grateful to Prof. Holger Hyd6n for his encouragement and generous support. This investigation was supported by the Swedish Medical Research Council and by the National Institute of Child Health and H u m a n Development No. 1 F2 HD-22, 166-01, U.S. Public Health Service. Institute of Neurobiology, Faculty of Medicine, University of Gothenburg, Gothenburg (Sweden)
ANANT R. DRAVID* THOMAS E. DUFFY**
* Present address: Division of Neurosciences, City of Hope National Medical Center, Duarte, Calif. 91010, U.S.A. * * Postdoctoral Fellow, U.S. Public Health Service. Present address: Department of Pharmacology, Washington University School of Medicine, St. Louis, Mo. 63110, U.S.A. Brain Research, 16 (1969) 516-519
SHORT COMMUNICATIONS
519
1 CHAMBON, P., RAMUZ, M., AND DOLY, J., Relation between soluble DNA-dependent R N A polymerase and 'aggregate' RNA polymerase, Biochem. biophys. Res. Commun., 21 (1965) 156-161. 2 CHAMBON, P., RAMUZ, M., MANDEL, P., AND DOLY, J,, The influence of ionic strength and a polyanion on transcription in vitro. I. Stimulation of the aggregate R N A polymerase from rat liver nuclei, Biochim. biophys. Acta (Amst.), 157 (19681 504-519. 3 DRAVID, A. R., DUFFY, T. E,, AND HAGL]D, K., Studies on in vitro R N A synthesis in human brain tumor nuclei, Brain Research, 12 (19691 2422.45. 4 FOCHS, E., MILLETTE, R. L., ZILLIG, W., AND WALTER, G., Influence of salts on RNA synthesis by DNA-dependent R N A polymerase from Escherichia co6, Europ. J. Biochem., 3 (1967) 183-193. 5 GOLDBERG, I. H.. Ribonucleic acid synthesis in nuclear extracts of mammalian cells grown in suspension culture: effect of ionic strength and surface active agents, Biochim. biophys. Acta (Amst.), 51 (1961) 201-204. 6 JOHNSON,J. D., JANT, B. A., SOKOLOFF, L., AND KAUFMAN,S., R N A polymerase in rat liver nuclei: Biphasic stimulation with ammonium sulphate, Biochim. biophys. Acta (Amst.), 179 (1969) 526-529. 7 KtSSANE, J. M., AND ROBBINS, E., The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. biol. Chem., 233 (1958) 184 188. 8 LOVT~UP-REIN, H., AND McEwEN, B. S., Isolation and fractionation of rat brain nuclei, J. Cell Biol., 30 (1966) 405-415. 9 RAMUZ, M., DOLY, J., MANDEL, P., AND CHAMBON, P,, A soluble DNA-dependent R N A polymerase in nuclei of non-dividing animal cells, Biochem. biophys. Res. Commun., 19 (1965) 114-120. 10 STERNER, D. F., AND KtNG, J., Insulin stimulated ribonucleic acid synthesis and R N A polymerase activity in alloxan diabetic rat liver, Biochim. biophys. Aeta (Amst.), 119 (1966) 510-516. I I WIDNELL, C. C., AND TATA, J. R., Studies on the stimulation by ammonium sulphate of the DNAdependent R N A polymerase of isolated rat liver nuclei, Biochhn. biophys. Acta (Amst.), 123 (1966) 478 492. (Accepted September 26th, 1969)
Brain Research, 16 (1969) 516-519
SHORT COMMUNICATIONS
Studies on in vitro RNA syn~sis in rat brain nuclei: biphasic stimulation of RNA polymerase with ammonium sulfate Stimulation of RNA polymerase (nucleoside triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) activity by ammonium sulfate has been observed with the 'aggregate' enzyme prepared from a variety of mammalian cell nuclei '~,s. This effect has been suggested to result from (a) unmasking of a latent R N A potymerase activity al, (b) dissociation of histones from the DNA template thus providing more D N A loci for transcription 1, or (c) inhibition of degradative enzymes 1°. A soluble DNA-dependent RNA polymerase activity in rat liver nuclei having metal and pH requirements similar to those of the purified bacterial RNA-polymerase has been reported 9. Contrary to the stimulatory effect of ammonium sulfate upon the 'aggregate' RNA polymerase, the soluble enzyme activity is strongly inhibited when ammonium sulfate is present in the incubation medium prior to enzyme addition 9. This communication describes a biphasic effect of ammonium sulfate on the RNA potymerase activity in rat brain nuclei. Preliminary studies on the enzyme activity in enriched neuronal and glial nuclei are also reported. Albino rats of Sprague-Dawley strain weighing about 200 g were used. The preparation of total brain nuclei was carried out as previously described ;~ except that the cerebellum was removed prior to homogenization. The fractionation of brain nuclei into 'large' (neuronal) and 'small' (glial) nuclei by sucrose density gradient centrifugation was performed by a modification of the method of Lovtrup and McEwenL A discontinuous gradient of 2.2, 2.4 and 2.6 M sucrose solutions was prepared and allowed to stand for I h in the cold. The suspension of brain nuclei in 2.0 M sucrose solution was layered over the gradient and centrifuged in a Spinco SW 25 rotor at 53,000 x g for 40 rain. Phase contrast examination of the band obtained at the interphase of 2.2 and 2.4 M sucrose showed a fairly uniform population of large, oval or round nuclei with one distinct nucleolus. The interphase between 2.4 and 2.6 M sucrose consisted chiefly of small nuclei with dense chromatin. These two nuclear populations were referred to as 'enriched' neuronal and glial nuclei, respectively. The conditions of the enzyme assay and the measurement of radioactivity were as described previously 3 except that the incubations were carried out for 10 rain. D N A was measured by the fluorometric method of Kissane and RobbinsL The effects of ammonium sulfate on RNA polymerase activity in whole brain nuclei in the presence of the optimum amount of MnCI2 (4 raM) are shown in Fig. 1. When the salt was added to the incubation mixture before initiation of R N A synthesis (lower curve) a slight stimulation was observed at low ammonium sulfate concentration and above 0.41 M the enzyme activity was inhibited. However, when ammonium sulfate was added to the complete incubation medium after RNA synthesis had already started (upper curve) a several-fold stimulation of the reaction was observed at an optimal salt concentration of 0.33 M. The rate decreased at higher ammonium sulfate concentration but even at 0.66 M a two-fold stimulation over the control persisted, l f t h e reaction were started by addition of enzyme to the complete incubation mixture containing ammonium sulfate, the stimulation of polymerase Brain Research,
16 (1969) 516-519
SHORT COMMUNICATIONS
5 17
1.5 /o,
/ ,, l o
,
\
J
........
;;S
0.50
0['
0.1
........~ . . . ....
0.2
0.5
ON
0.5
",~
0.6
0.7
0.8
M (NH4) z S04
Fig. I. The incorporation of [aH]CTP by the aggregate R N A polymerase from rat brain nuclei as a function of (NH4)eSO4 concentration. The incubation medium contained (in ffmoles) Tris-HCl, pH 8.1, 40; MnCI~, 1; KCI, 15; NaF, 5; ATP, GTP and UTP, 0.25 each; [aH]CTP (sp. activity, 40 ffC/ffmole) 0.125 and enzyme in a final volume of 0.25 ml. Incubation at 37 ° for 10 min. Addition of (NH4)2804, • - • before initiation of RNA synthesis and C- - -(3 after initiation. × . . . x, Reaction started by addition of the enzyme to complete incubation medium including (NH4)2504. 0.75
E
/
0.50-
o
g
<
x, 0.25 -
o=
o
oi~
d2
ds
o)
o.'s
o:~
o}
M (NH4) 2 S04
Fig. 2. The incorporation of [aH]CTP by the aggregate R N A polymerase from rat brain and liver nuclei as a function of (NHa)2SO4 concentration. (NH4)zSO4 was added after initiation of R N A synthesis. For incubation conditions see legend to Fig. 1. 2. - f,, Brain nuclear enzyme and • •, liver nuclear enzyme.
a c t i v i t y was i n t e r m e d i a t e (Fig. I, m i d d l e curve). In this case also, no i n h i b i t i o n o f a c t i v i t y c o u l d be d e m o n s t r a t e d 1.0 M ( n o t shown).
with a m m o n i u m
sulfate c o n c e n t r a t i o n s as high as
T h e effects o f a m m o n i u m sulfate u p o n the ' a g g r e g a t e ' R N A p o l y m e r a s e a c t i v i t y in brain nuclei are q u a l i t a t i v e l y s i m i l a r to t h o s e o b s e r v e d with purified b a c t e r i a l R N A p o l y m e r a s e 4. M o r e o v e r , u n l i k e liver nuclei '~ the s t i m u l a t i o n o f R N A p o l y m e r a s e in brain nuclei was c o n s i d e r a b l y h i g h e r w h e n the salt was a d d e d a f t e r i n i t i a t i o n o f R N A synthesis t h a n w h e n a m m o n i u m
sulfate was p r e s e n t p r i o r to i n i t i a t i o n . Brain Research, 16 (1969) 516-519
518
SHORT COMMUNICATIONS
TABLE I OF [3H]UTP BY T H E (/,#moles UMP/mg DNA/IO rain)
INCORPORATION
AGGREGATE
RNA
P O L Y M E R A S E F R O M R A T B R A I N N U C I El
Incubation conditions were as described in legend to Fig. 1, except that [3H]CTP was replaced by [sH]UTP.
Complete incubation medium Omit ATP, GTP and CTP Complete ÷ (NH4)~SO4
Unfractionated nuclei
Neuronal nuclei
Glial mtclel
215.2 101.0 1505.4
227.5 88.3 2270.0
160.2 97.7 96 I. 2
Further studies with a finer a m m o n i u m sulfate concentration gradient revealed a biphasic stimulation of R N A polymerase activity in brain nuclei. The results of these studies are shown in Fig. 2. The upper curve depicts the response of the brain nuclear enzyme activity to the addition of a m m o n i u m sulfate after initiation of R N A synthesis. When liver nuclei were incubated under identical conditions a broad peak of stimulation was observed (Fig. 2, lower curve). Regardless of the mechanism of stimulation, brain and liver nuclear RN A polymerase activities respond differentially to a m m o n i u m sulfate under identical conditions of incubation. Under somewhat different incubation conditions than those used in the present investigation, a biphasic stimulation of the liver enzyme activity by a m m o n i u m sulfate has recently been reported 6. Preliminary results of the R N A polymerase activity in brain nuclei (unfractionated, neuronal and glial) are summarized in Table I. A m m o n i u m sulfate was found to increase the incorporation of labeled U T P by all 3 fractions, although the effect appeared to be most marked in the neuronal nuclei. In addition, the incorporation of [zH]UTP in the absence of ATP, CTP and G T P was considerably higher in glial nuclei (61 ~o of that found in the presence of the other 3 nucleoside triphosphates) as compared with neuronal nuclei (39 ~o). Whether the incorporation of labeled U T P in the absence of ATP, G T P and CTP is due to polyuridylate synthesis is uncertain at present. However, under similar conditions of incubation we have observed a high rate of poly U synthesis in human brain tumors of glial origin 3. The authors are grateful to Prof. Holger Hyd6n for his encouragement and generous support. This investigation was supported by the Swedish Medical Research Council and by the National Institute of Child Health and H u m a n Development No. 1 F2 HD-22, 166-01, U.S. Public Health Service. Institute of Neurobiology, Faculty of Medicine, University of Gothenburg, Gothenburg (Sweden)
ANANT R. DRAVID* THOMAS E. DUFFY**
* Present address: Division of Neurosciences, City of Hope National Medical Center, Duarte, Calif. 91010, U.S.A. * * Postdoctoral Fellow, U.S. Public Health Service. Present address: Department of Pharmacology, Washington University School of Medicine, St. Louis, Mo. 63110, U.S.A. Brain Research, 16 (1969) 516-519
SHORT COMMUNICATIONS
519
1 CHAMBON, P., RAMUZ, M., AND DOLY, J., Relation between soluble DNA-dependent R N A polymerase and 'aggregate' RNA polymerase, Biochem. biophys. Res. Commun., 21 (1965) 156-161. 2 CHAMBON, P., RAMUZ, M., MANDEL, P., AND DOLY, J,, The influence of ionic strength and a polyanion on transcription in vitro. I. Stimulation of the aggregate R N A polymerase from rat liver nuclei, Biochim. biophys. Acta (Amst.), 157 (19681 504-519. 3 DRAVID, A. R., DUFFY, T. E,, AND HAGL]D, K., Studies on in vitro R N A synthesis in human brain tumor nuclei, Brain Research, 12 (19691 2422.45. 4 FOCHS, E., MILLETTE, R. L., ZILLIG, W., AND WALTER, G., Influence of salts on RNA synthesis by DNA-dependent R N A polymerase from Escherichia co6, Europ. J. Biochem., 3 (1967) 183-193. 5 GOLDBERG, I. H.. Ribonucleic acid synthesis in nuclear extracts of mammalian cells grown in suspension culture: effect of ionic strength and surface active agents, Biochim. biophys. Acta (Amst.), 51 (1961) 201-204. 6 JOHNSON,J. D., JANT, B. A., SOKOLOFF, L., AND KAUFMAN,S., R N A polymerase in rat liver nuclei: Biphasic stimulation with ammonium sulphate, Biochim. biophys. Acta (Amst.), 179 (1969) 526-529. 7 KtSSANE, J. M., AND ROBBINS, E., The fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system, J. biol. Chem., 233 (1958) 184 188. 8 LOVT~UP-REIN, H., AND McEwEN, B. S., Isolation and fractionation of rat brain nuclei, J. Cell Biol., 30 (1966) 405-415. 9 RAMUZ, M., DOLY, J., MANDEL, P., AND CHAMBON, P,, A soluble DNA-dependent R N A polymerase in nuclei of non-dividing animal cells, Biochem. biophys. Res. Commun., 19 (1965) 114-120. 10 STERNER, D. F., AND KtNG, J., Insulin stimulated ribonucleic acid synthesis and R N A polymerase activity in alloxan diabetic rat liver, Biochim. biophys. Aeta (Amst.), 119 (1966) 510-516. I I WIDNELL, C. C., AND TATA, J. R., Studies on the stimulation by ammonium sulphate of the DNAdependent R N A polymerase of isolated rat liver nuclei, Biochhn. biophys. Acta (Amst.), 123 (1966) 478 492. (Accepted September 26th, 1969)
Brain Research, 16 (1969) 516-519