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Synthesis of ribonucleic acids in the adrenal cortex: Eatly effects of adrenocorticotropic hormone
I~O
PRELIMINARY
NOTES
PN 91023
Synthesis of ribonucleic acids in the adrenal cortex: Early effects of adrenocorticotropic hormone We were interested in studying the early effects of the administration of ACTH on the formation of rapidly labeled RNA fractions in adrenals. The intraperitoneal injection of 133/~g of Wilson ACTH (2o units) into each of 8 male albino guinea-pigs (4oo-5oo g) was followed I h later by that of 4 mC Ea2p]pI, carrier-free, and 35o uC E5-3HJuridine (II 8oo#C/mmole). After 3ornin, the animals were killed and the adrenals trimmed, demedullated partially, diced, and ground in a glass tissue-grinder in an aqueous medium (IO ml per g tissue) having the following composition: 32o mM sucrose, I m ~ CaCI~, 50 m ~ KC1, i mIV[5IgC12, and 50 mM Tris-I-IC1 buffer (pH 7.5)The filtrate through cheese cloth, centrifuged at 6 o o × g for io rain, yielded the supernatant fluid, containing the cytoplasmic fraction, and the nuclear pellet. The latter was washed with 0.5 o/ /o Triton X-Ioo (ref. I), 0.5 mM CaC12, 0.5 mM MgCI~, and IO rnM monosodium maleate-NaOH buffer (pH 6.o), and sedimented through 2 IV[ sucrose at 35 ooo ×g. The nuclear pellet and the material precipitated from the cytoplasmic supernatant fluid ~dth cold 5 o//otrichloroacetic acid, both washed with cold 5 070/ trichloroacetic acid-o.I M Nati2PO 4, acetone, and CI-ICla-CH30I-[ (2 : I, v/v), were treated twice (IOO°, 30 rain) with io % LiC1 (pH 7.0) to extract the nucleic acids. These were precipitated with 2 volumes of ethanol and their solutions streaked on filter TABLE SPECIFIC
I
N U C L E A R A N D CYTOPLASMIC R N A OF 6UINEA-PIG A D R E N A L CORTEX WITH AND WITHOUT A C T H ADMINISTRATION
RADIOACTIVITY
OF
T h e t w o a d r e n a l s per a n i m a l w e i g h e d 1 5 o - 2 o o r a g . T h e m e a n c o n t e n t s of p a r t i a l l y d e m e d u l l a t e d g l a n d s w e r e per IOO mg: D N A , 9 5 / z g ; t o t a l R N A 1 9 8 / * g ; c y t o p l a s m i c R N A , 1 7 6 / z g ; n u c l e a r R N A , 2 2 / * g . T h e specific a c t i v i t y v a l u e s are t h e m e a n of d e t e r m i n a t i o n s o n pairs of a d r e n a l s from four s e p a r a t e a n i m a l s , To t h e controls, n o A C T H w a s a d m i n i s t e r e d . V a l u e s are g i v e n as c o u n t s / r a i n per I o o / z g R N A . P is t h e p r o b a b i l i t y t h a t t h e m e a n s are n o t different. N . S . = n o t s i g n i f i c a n t . See t e x t for o t h e r details.
alter precursor in/ection
Time RNA
(min)
[aH] Uridine
Control
A CTH
~,azp] p i P
Control
,4 C T H
P
Total RNA
3° 60 240
37 84 322
I57 223 400
37 81 I304
66 878 5711
Nuclear RNA
3° 60 240
28 90 692
lO6 272 513
34 238 2166
56 825 2862
<0.05
Cytoplasmic RNA
3° 60 240
3° lO3 2o3
59 195 291
48 66 1475
43 165 4490
N.S.
Biochim. Biophys. Acta, 91 (1964) 1 8 o - 1 8 2
PRELIMINARY
NOTES
I8~
paper and washed ~ by irrigation with buffered ammonium isobutyrate solvent. After aqueous extraction and determination of the DNA and RNA contents, the remainder was reprecipitated (5 % trichloroacetic acid, --IO °, overnight), collected TABLE
II
COMPOSITION OF NUCLEIC ACIDS OF GUINEA-PIG AND DISTRIBUTION OF RADIOACTIVITY" AMONG (2'+3')-NUCLEOTIDE CONSTITUENTS OF ~2P-LABELED R N A A, G, C, U d e n o t e a d e n y l i c , g u a n y l i c , c y t i d y l i c , a n d u r i d y l i c a c i d s i n R N A ; A, G, C, T s t a n d for a d e n i n e , g u a n i n e , c y t o s i n e , a n d t h y m i n e i n D N A . T h e c o m p o s i t i o n of D N A a n d R N A is e x p r e s s e d as m o l e s of c o n s t i t u e n t p e r i o o g r a m a t o m s of n u c l e i c a c i d P ; t h a t of t h e p u l s e - l a b e l e d R N A as % of "eP i n c o r p o r a t e d i n t o R N A 3 ° r a i n a f t e r i n j e c t i o n of i s o t o p e . See t e x t for d e t a i l s .
A
G
C
U(T)
29.8
21.7
20.4
28.6
Total R N A
19.7
32.7
27-1
20.5
Nuclear R N A
2o.~
27.6
30.8
21. 5
P u l s e - R N A (control)
28.0
24.9
18.3
28.8
P u l s e - R N A (ACTH)
20. 3
23.9
27.5
28.3
Nucleotide constituents
DNA
~) NUCLEAR
2o0~ ~5 ®"UcLEA"
+
05
O 0.4 £M
I
E
A
5o ~
4o~
150~
w
-
,ooi
,~' '
0'2
o
o, : ~ '~:o~ ; +.° 03
@
05 O] 09 II 13 t5 - NeCI MOLARITY OF ELUATE
50 OC
w
],t
o~0.2 CO
Q3
CYTOPLASMIC
•
~:"~+,., , ~,, 05
0.7 0.9 I.I L3 [ 5 - 1 5 NeCt MOLARITY OF ELUATE
to
25 E:
++ +o+o.+
40 ,~u >-
o 0,3 Z
30
_;
.,o ~
02
20
w
~ o,s
~5_~
~ 0.4 •
~o~
0.7 0.9 I.I [3 L5- - I . 5 NaCt MOLARITY OF ELUATE
I0 ~
(~) GYTOPLASMIC / ' ~
5o "~
004
03
5
2o g
~,, : I
*~ 0,1
15
E•.O,5
3O
:~
z0°.3
\
9
,oo
s~
'¢0,2 0.3
Q5
0.7 0.9 I.I [3 I~ - - I . 5 NaCt MOLARITY OF ELUATE
Fig. I. Fractionation on columns of m e t h y l a t e d serum a l b u m i n - k i e s e l g u h r of nuclear and cytoplasmic R N A of adrenal cortex isolated from guinea-pigs, with and w i t h o u t p r e t r e a t m e n t with ACTH, after an exposure to isotopic precursors for 3 ° rain. R N A was extracted with sodium dodecyl s u l f a t e - a q u e o u s phenol and before fractionation mixed with carrier ]RNA isolated from guinea-pig liver. Elation with a linear gradient of o.3-1. 5 M NaC1 in 0.025 M Tris-HC1 (pH 7.0): 4-S RNA, 0.4 M; D N A and a c c o m p a n y i n g RNA, 0. 5 M; 2o-S RNA, 0. 7 M; 3o-S RNA, 0.8 M; " h e a v y " RNA, above I.I M. Solid lines represent ultraviolet absorbance; broken lines indicate radioactivity, with full circles representing the animals treated with ACTH, and open circles the controls. A: controls receiving [32pjpt, ACTH-animals [3H]uridine; B: reverse a r r a n g e m e n t of precursors. The second of the three radioactivity peaks in the region of cytoplasmic 3o-S R N A is designated " b " .
Biochim. Biophys. Acta, 91 (1964) 18o-182
182
PRELIMINARY NOTES
on membrane filters (Schleicher and Schiill, B 6), and analyzed for radioactivity by liquid-scintillation counting. The filters were then dissolved in acetone, and RNA was collected and hydrolysed with o.3 N KOH. The hydrolysate was applied to filter paper, desalted by irrigation with n-butanol (saturated with H20), and subiected to electrophoresis in 2o c!~ acetic acid, adjusted to pIK 3.1 with ammonia, at 20 V/cm. The nucleotides were eluted with o.I M potassium phosphate (pH 7.o) and their radioactivities determined on planchets by gas-flow counting. The effect of ACTH on RNA synthesis, significant in most inslances, is shown in Table I which includes results of longer periods of isotope incorporation. Table II summarizes, in addition to the nucleotide composition of DNA, total RNA, and nuclear RNA, data on the distribution of a"P among the (2' +3')-nucleotides released by the hydrolysis of the RNA formed during an exposure of 3o rain to the isotope. Here, again, the effect of ACTH is evident. Experiments on the elution behavior of nuclear and cytoplasmic RNA, labeled by a a2p pulse of 3o rain, on a column of methylated serum albumin-kieselguhr are shown in Fig. I. These experiments were always performed in two series: (A) the animals treated with ACTH received EaH~uridine, the controls I32P~PI; (B) the ACTH animals received radiophosphate, tile controls labeled uridine. ACTH is seen to exert a generally stimulatory effect on the synthesis of nuclear RNA, most conspicuously in the fractions eluted after ribosomal RNA. In the preparations of cytoplasmic RNA the changes appear mostly in the 3o-S fraction. An outstanding peak of radioactivity, labeled "b", is seen consistently in specimens labeled by exposure to isotope for 3o-9o min. A full account of this and related work, which has been supported by research grants from the National Institutes of Health, U.S. Public Health Service, and the American Cancer Society, will be published later. E.D.B. was a Postdoctoral Research Fellow of the American Cancer Society, I962-64 .
Cell Chemistry Laboratory, Department o/ Biochemistry, College o/ Physicians and Surgeons, Columbia University, New York, N.Y. (U.S.A.)
EDWIN D. BRANSOME, JR.* t~RWIN CHARGAFF
1 W. C. HYMER, Federation Proc., 2i (1963) lO 4. 2 T. D. PRICE, ]-1. A. HINDS AND R. S. BROWN, J. Biol. Chem., 238 (1963) 31I.
Received July I3th, 1963 * P r e s e n t address: Division of Endrocrinology, Scripps Clinic and Research F o u n d a t i o n , L a Jolla, Calif. (U.S.A.).
Bioehim. Biophys. Acta, 91 (1964) 18o--i82
PRELIMINARY
NOTES
PN 91023
Synthesis of ribonucleic acids in the adrenal cortex: Early effects of adrenocorticotropic hormone We were interested in studying the early effects of the administration of ACTH on the formation of rapidly labeled RNA fractions in adrenals. The intraperitoneal injection of 133/~g of Wilson ACTH (2o units) into each of 8 male albino guinea-pigs (4oo-5oo g) was followed I h later by that of 4 mC Ea2p]pI, carrier-free, and 35o uC E5-3HJuridine (II 8oo#C/mmole). After 3ornin, the animals were killed and the adrenals trimmed, demedullated partially, diced, and ground in a glass tissue-grinder in an aqueous medium (IO ml per g tissue) having the following composition: 32o mM sucrose, I m ~ CaCI~, 50 m ~ KC1, i mIV[5IgC12, and 50 mM Tris-I-IC1 buffer (pH 7.5)The filtrate through cheese cloth, centrifuged at 6 o o × g for io rain, yielded the supernatant fluid, containing the cytoplasmic fraction, and the nuclear pellet. The latter was washed with 0.5 o/ /o Triton X-Ioo (ref. I), 0.5 mM CaC12, 0.5 mM MgCI~, and IO rnM monosodium maleate-NaOH buffer (pH 6.o), and sedimented through 2 IV[ sucrose at 35 ooo ×g. The nuclear pellet and the material precipitated from the cytoplasmic supernatant fluid ~dth cold 5 o//otrichloroacetic acid, both washed with cold 5 070/ trichloroacetic acid-o.I M Nati2PO 4, acetone, and CI-ICla-CH30I-[ (2 : I, v/v), were treated twice (IOO°, 30 rain) with io % LiC1 (pH 7.0) to extract the nucleic acids. These were precipitated with 2 volumes of ethanol and their solutions streaked on filter TABLE SPECIFIC
I
N U C L E A R A N D CYTOPLASMIC R N A OF 6UINEA-PIG A D R E N A L CORTEX WITH AND WITHOUT A C T H ADMINISTRATION
RADIOACTIVITY
OF
T h e t w o a d r e n a l s per a n i m a l w e i g h e d 1 5 o - 2 o o r a g . T h e m e a n c o n t e n t s of p a r t i a l l y d e m e d u l l a t e d g l a n d s w e r e per IOO mg: D N A , 9 5 / z g ; t o t a l R N A 1 9 8 / * g ; c y t o p l a s m i c R N A , 1 7 6 / z g ; n u c l e a r R N A , 2 2 / * g . T h e specific a c t i v i t y v a l u e s are t h e m e a n of d e t e r m i n a t i o n s o n pairs of a d r e n a l s from four s e p a r a t e a n i m a l s , To t h e controls, n o A C T H w a s a d m i n i s t e r e d . V a l u e s are g i v e n as c o u n t s / r a i n per I o o / z g R N A . P is t h e p r o b a b i l i t y t h a t t h e m e a n s are n o t different. N . S . = n o t s i g n i f i c a n t . See t e x t for o t h e r details.
alter precursor in/ection
Time RNA
(min)
[aH] Uridine
Control
A CTH
~,azp] p i P
Control
,4 C T H
P
Total RNA
3° 60 240
37 84 322
I57 223 400
37 81 I304
66 878 5711
Nuclear RNA
3° 60 240
28 90 692
lO6 272 513
34 238 2166
56 825 2862
<0.05
Cytoplasmic RNA
3° 60 240
3° lO3 2o3
59 195 291
48 66 1475
43 165 4490
N.S.
Biochim. Biophys. Acta, 91 (1964) 1 8 o - 1 8 2
PRELIMINARY
NOTES
I8~
paper and washed ~ by irrigation with buffered ammonium isobutyrate solvent. After aqueous extraction and determination of the DNA and RNA contents, the remainder was reprecipitated (5 % trichloroacetic acid, --IO °, overnight), collected TABLE
II
COMPOSITION OF NUCLEIC ACIDS OF GUINEA-PIG AND DISTRIBUTION OF RADIOACTIVITY" AMONG (2'+3')-NUCLEOTIDE CONSTITUENTS OF ~2P-LABELED R N A A, G, C, U d e n o t e a d e n y l i c , g u a n y l i c , c y t i d y l i c , a n d u r i d y l i c a c i d s i n R N A ; A, G, C, T s t a n d for a d e n i n e , g u a n i n e , c y t o s i n e , a n d t h y m i n e i n D N A . T h e c o m p o s i t i o n of D N A a n d R N A is e x p r e s s e d as m o l e s of c o n s t i t u e n t p e r i o o g r a m a t o m s of n u c l e i c a c i d P ; t h a t of t h e p u l s e - l a b e l e d R N A as % of "eP i n c o r p o r a t e d i n t o R N A 3 ° r a i n a f t e r i n j e c t i o n of i s o t o p e . See t e x t for d e t a i l s .
A
G
C
U(T)
29.8
21.7
20.4
28.6
Total R N A
19.7
32.7
27-1
20.5
Nuclear R N A
2o.~
27.6
30.8
21. 5
P u l s e - R N A (control)
28.0
24.9
18.3
28.8
P u l s e - R N A (ACTH)
20. 3
23.9
27.5
28.3
Nucleotide constituents
DNA
~) NUCLEAR
2o0~ ~5 ®"UcLEA"
+
05
O 0.4 £M
I
E
A
5o ~
4o~
150~
w
-
,ooi
,~' '
0'2
o
o, : ~ '~:o~ ; +.° 03
@
05 O] 09 II 13 t5 - NeCI MOLARITY OF ELUATE
50 OC
w
],t
o~0.2 CO
Q3
CYTOPLASMIC
•
~:"~+,., , ~,, 05
0.7 0.9 I.I L3 [ 5 - 1 5 NeCt MOLARITY OF ELUATE
to
25 E:
++ +o+o.+
40 ,~u >-
o 0,3 Z
30
_;
.,o ~
02
20
w
~ o,s
~5_~
~ 0.4 •
~o~
0.7 0.9 I.I [3 L5- - I . 5 NaCt MOLARITY OF ELUATE
I0 ~
(~) GYTOPLASMIC / ' ~
5o "~
004
03
5
2o g
~,, : I
*~ 0,1
15
E•.O,5
3O
:~
z0°.3
\
9
,oo
s~
'¢0,2 0.3
Q5
0.7 0.9 I.I [3 I~ - - I . 5 NaCt MOLARITY OF ELUATE
Fig. I. Fractionation on columns of m e t h y l a t e d serum a l b u m i n - k i e s e l g u h r of nuclear and cytoplasmic R N A of adrenal cortex isolated from guinea-pigs, with and w i t h o u t p r e t r e a t m e n t with ACTH, after an exposure to isotopic precursors for 3 ° rain. R N A was extracted with sodium dodecyl s u l f a t e - a q u e o u s phenol and before fractionation mixed with carrier ]RNA isolated from guinea-pig liver. Elation with a linear gradient of o.3-1. 5 M NaC1 in 0.025 M Tris-HC1 (pH 7.0): 4-S RNA, 0.4 M; D N A and a c c o m p a n y i n g RNA, 0. 5 M; 2o-S RNA, 0. 7 M; 3o-S RNA, 0.8 M; " h e a v y " RNA, above I.I M. Solid lines represent ultraviolet absorbance; broken lines indicate radioactivity, with full circles representing the animals treated with ACTH, and open circles the controls. A: controls receiving [32pjpt, ACTH-animals [3H]uridine; B: reverse a r r a n g e m e n t of precursors. The second of the three radioactivity peaks in the region of cytoplasmic 3o-S R N A is designated " b " .
Biochim. Biophys. Acta, 91 (1964) 18o-182
182
PRELIMINARY NOTES
on membrane filters (Schleicher and Schiill, B 6), and analyzed for radioactivity by liquid-scintillation counting. The filters were then dissolved in acetone, and RNA was collected and hydrolysed with o.3 N KOH. The hydrolysate was applied to filter paper, desalted by irrigation with n-butanol (saturated with H20), and subiected to electrophoresis in 2o c!~ acetic acid, adjusted to pIK 3.1 with ammonia, at 20 V/cm. The nucleotides were eluted with o.I M potassium phosphate (pH 7.o) and their radioactivities determined on planchets by gas-flow counting. The effect of ACTH on RNA synthesis, significant in most inslances, is shown in Table I which includes results of longer periods of isotope incorporation. Table II summarizes, in addition to the nucleotide composition of DNA, total RNA, and nuclear RNA, data on the distribution of a"P among the (2' +3')-nucleotides released by the hydrolysis of the RNA formed during an exposure of 3o rain to the isotope. Here, again, the effect of ACTH is evident. Experiments on the elution behavior of nuclear and cytoplasmic RNA, labeled by a a2p pulse of 3o rain, on a column of methylated serum albumin-kieselguhr are shown in Fig. I. These experiments were always performed in two series: (A) the animals treated with ACTH received EaH~uridine, the controls I32P~PI; (B) the ACTH animals received radiophosphate, tile controls labeled uridine. ACTH is seen to exert a generally stimulatory effect on the synthesis of nuclear RNA, most conspicuously in the fractions eluted after ribosomal RNA. In the preparations of cytoplasmic RNA the changes appear mostly in the 3o-S fraction. An outstanding peak of radioactivity, labeled "b", is seen consistently in specimens labeled by exposure to isotope for 3o-9o min. A full account of this and related work, which has been supported by research grants from the National Institutes of Health, U.S. Public Health Service, and the American Cancer Society, will be published later. E.D.B. was a Postdoctoral Research Fellow of the American Cancer Society, I962-64 .
Cell Chemistry Laboratory, Department o/ Biochemistry, College o/ Physicians and Surgeons, Columbia University, New York, N.Y. (U.S.A.)
EDWIN D. BRANSOME, JR.* t~RWIN CHARGAFF
1 W. C. HYMER, Federation Proc., 2i (1963) lO 4. 2 T. D. PRICE, ]-1. A. HINDS AND R. S. BROWN, J. Biol. Chem., 238 (1963) 31I.
Received July I3th, 1963 * P r e s e n t address: Division of Endrocrinology, Scripps Clinic and Research F o u n d a t i o n , L a Jolla, Calif. (U.S.A.).
Bioehim. Biophys. Acta, 91 (1964) 18o--i82