- Email: [email protected]
Estrogen stimulation of synthesis of specific proteins and RNA polymerase activity in the immature chick oviduct
204
PRELIMINARY NOTES
being 7o-S ribosomes '6. (d) By short-time labelling of RNA with a2Pl, and protein with EaH~lysine, a large percentage of each trichloroacetic acid-precipitable label was found in Peak II. Thus, functional polysomes appear to be associated with the membrane fraction of the cell in phage-infected as well as in normal E. coll. i am grateful to Dr. A. P. NYGAARD and Dr. J. G. HAUGE for support and helpful discussion of this work.
Department o/Biochemistry, University o/Bergen, Bergen (Norway) I 2 3 4 5 6 7 8 9 IO Ii 12 I3 14 15 16
LARS H A A R R
S. P. CHAMPE,Ann. Rev. Microbiol., 17 (1963) 87. M. SEKIGUCHI AND S. S. COHEN, J. Mol. Biol., 8 (1964) 638. 1R. B. KHESIN AND M. F. SHEMYAKIN, Biokhimiya, 27 (1962) 761. B. D. HALL, A. P. NYGAARD AND M. H. GREEN, J. Mol. Biol., 9 (1964) 143. K. F. BAUTZ, T. I~ASAI, E. REILI.Y AN]) F. i . BAUTZ, .Proc. Natl. Acad. Sci. U. S., 55 (1966) lO81. E. P. GEIDUSCHEK, L. SNYDER, A. J. E. COLVILL AND M. SARNAT, J. Mol. Biol., 19 (1966) 541 . J. C. SuiT, Biochirn. Biophys. dcta, 72 (1963) 488. D. SCttLESSlNGER, J. 3/Iol. Biol., 7 (1963) 569. P. A. HALLBERG AND J. G. HAUGE, Biochim. Biophys. Acta, 95 (1965) 80. L. D. MOORE AND W. W. UMBREIT, Biochim. Biophys. Acta, lO 3 (1965) 466. A. P. NYGAARD AND B. D. HALL, J. Mol. Biol., 9 (1964) 125. J. c. SUIT, J. Bacteriol., 84 (1962) lO61. A. P. NYGAARD AND B. D. HALL, Biochem. Biophys. Res. Commun., 42 (1963) 98. L. GROSSMAN, S. S. LEVlNE AND W . S. ALLISON, J. Mol. Biol., 3 (1961) 47. R. W. HENDLER AND J. TANI, Biochim. Biophys. Acta, 80 (1964) 294. G. MANGIAROTTI AND D. SCHLESSlNGER, J. Mol. Biol., 20 (1966) 123.
Received April i9th, 1967 Biochim. Biophys. Acta, 145 (I967) 2o2-2o4
BBA 91176
Estrogen stimulation of synthesis of specific activity in the immature chick oviduct
proteins and R N A polymerase
The precise mechanism of action of estrogen in the stimulation of growth of estrogen-sensitive tissues such as the uterus is unresolved, but there is good evidence for an early stimulation of RNA and protein synthesis 1-s. Estrogens have been reported to induce increased levels of m a n y enzymes in the uterus 4 and to stimulate incorporation of Iz4Cjglycine into protein, [aH~cytidine into RNA, and a~Pi, El*Cjacetate, and I14Clglucose into lipid, in this organ 5. GORSKIe has also demonstrated stimulation of rat uterus RNA polymerase after estrogen administration in vivo. Estrogens stimulate oviduct growth in newborn and older chicksL Formation of secretion granules within the oviduct glands and release of an albumin-like material into the lumen of the chick oviduct were reported to follow administration of estrogen and progesterone or estrogen and androgen combinationsL Avidin, the biotin-binding egg-white protein, has been noted to appear in the estrogen-stimulated chick oviduct Biochim. Biophys. Acta, 145 (1967) 2o4-2o7
PRELIMINARY NOTES
205
after a single dose of progesterone (HERTZ 8, KORENMAN AND O'MALLEy9). This communication deals with the changes in the oviduct proteins, ovalbumin and lysozyme, oviduct RNA polymerase activity, and the ability to synthesize avidin in response to progesterone after in vivo administration of diethylstilbestrol to immature chicks. Four-day-old female Rhode Island Red chicks were iniected subcutaneously with 5 mg diethylstilbestrol dissolved in sesame oil daily for 15 days. At the indicated times the animals were sacrificed. The oviducts were removed, weighed, homogenized, and an aliquot of the lO5 ooo ×g supernatant was analyzed for ovalbumin, lysozyme or avidin. Ova]bumin was precipitated b y specific antibody 1° and quantified b y protein estimation of the precipitate using the FOLIN--LowRY procedure 1°. Agar-gel diffusion studies on Ouchterlony plates demonstrated that the precipitating protein was identica] with ovalbumin. Lysozyme was assayed by the method of LITWACKn. Avidin was measured using an assay involving specific binding of the avidin to D-[carboxyl-14Clbiotin, adsorption of the 14C-labeled avidin-biotin complex onto bentonite, and trapping the complex on a Millipore filter 12. Measurements of the various proteins are expressed per g oviduct for convenience, as calculations on the basis of mg protein b y the method of LOWRY13 gave essentially the same results. Nuclear RNA polymerase was measured in vitro on nuclei isolated from oviducts at various times following a single dose of diethylstilbestrol. Oviducts were homogenized in buffer containing 0.32 M sucrose, o.ooi M MgC] 2, 0.0004 M KH2PO 4, 0.0004 M K2HPO 4, p H 6.7, and filtered through single-napp flannelette, washed with homogenizing buffer (three times) and centrifuged for 9 ° min at 5 ° ooo ×g in a solution containing 2.2 M sucrose, o.ooi M MgC12, 0.0035 M K2HPO4, 0.00o 7 M ATP, p H 6. 7. The nuclei were then resuspended in 0.05 M Tris-HC1, 30 % (v/v) glycerol, and o.ooi M inercaptoethanol, p H 7.6. The assay involves incorporation of labeled nucleotide into an acid-insoluble ribonuclease-sensitive product, and is dependent on exogenous nucleotides and is inhibited by deoxyribonuclease and actinomycin D. Details of the isolation method will be reported e]sewhere (W. L. McGUIRE AND B. W. O'MALLEY, unpublished observations). 3°°° I
DeS
]
I00-
8
IOOO
8 I
0
3
6
9
J2
J5 DAYS
18
21
24
27
0
~
e
9
12 r5 DAYS
18
2~
24
2Y
Fig. I. Effect of diethylstilbestrol (DES) a d m i n i s t r a t i o n on o v a l b u m i n levels in the i m m a t u r e chick oviduct. E a c h chick received 5 mg diethylstilbestrol in oil (subcutaneously) daily for 15 days. Chicks were sacrificed at the days indicated and o v a l b u m i n d e t e r m i n a t i o n s were carried o u t on the o v i d u c t s as described in the text. E a c h p o i n t r e p r e s e n t s the m e a n of 4 animals in a representative experiment. Fig. 2. Effect of diethylstilbestrol (DES) a d m i n i s t r a t i o n on lysozyme levels in the i m m a t u r e chick oviduct. Protocol is exactly as in Fig. I.
Biochim. Biophys. Acta, 145 (1967) 2o4-2o 7
206
PRELIMINARY NOTES
The effect of diethylstilbestrol administration on ovalbumin levels in the immature (unstimulated) chick oviduct is shown in Fig. I. The increase is minimal over the first 6 days. During this period morphological changes in cell differentiatien are taking place with appearance and proliferation of epithelial glands thought to be involved in ovalbumin synthesis (P. O. KOHLER AND B. W. O ' M A L L E Y , unpublished observations). Induction of ovalbumin synthesis is striking between 6 and 15 days. The level of ovalbumin at day 15 is 300 times the initial baseline concentration. When diethylstilbestrol is discontinued, a 62 °/o decrease in levels of this oviduct protein is observed over the next I I days. The fall probably represents a combination of decreased synthesis, degradation, and secretory loss into the gland lumen. A change in total oviduct mass occurs from 5 ~o mg in the immature chick to 1. 5 to 3 g after 3 weeks of estrogen treatment. We know of no other tissue capable of such a marked growth response to a hormone. A similar induction of oviduct lysozyme synthesis occurs over the same period of diethylstilbestrol treatment (Fig. 2). Withdrawal of the hormone again leads to a 65 % fall in specific activity of the enzyme over the next I I days. Indaetion of avidin synthesis in the chick oviduct after administration of progesterone in vivo will be presented in detail elsewhere 9. However, we have examined the role that diethylstilbestrol administration plays in the ability of the oviduct to respond to a progestational stimulus (Fig. 3). Progesterone-induced avidin synthesis is low in the hor-
25
2o
0
I0 OAYS OFOES(5mg)
t5
Fig. 3- E f f e c t of d i e t h y l s t i l b e s t r o l (DES) a d m i n i s t r a t i o n on t h e chick o v i d u c t c a p a c i t y to s y n t h e s i z e avidin. D i e t h y l s t i l b e s t r o l w a s a d m i n i s t e r e d as in Fig. I. A single 5-rag injection of p r o g e s t e r o n e in p r o p y l e n e glycol ( s u b c u t a n e o u s l y ) w a s a d m i n i s t e r e d 24 h prior to sacrificing t h e a n i m a l s . Avidin ( m e a n of io a n i m a l s w i t h range) c o n t e n t w a s m e a s u r e d in each o v i d u c t as described in t h e t e x t a n d is r e p r e s e n t e d b y t h e b a r g r a p h . T h e e x p e r i m e n t w a s r e p e a t e d twice. No m e a s u r a b l e a v i d i n is p r e s e n t in o v i d u c t s of d i e t h y l s t i l b e s t r o l - t r e a t e d chicks w h i c h h a v e n o t received progesterone.
monally immature oviduct (day o) but the capacity for avidin synthesis increases a great deal by d a y 6 of diethylstilbestrol administration. Further estrogen reduces the capacity of the oviduct to synthesize avidin. If the diethylstilbestrol treatment is extended beyond 21 days, only 1.5-4.o #g avidin/g oviduct is found after induction by progesterone. Estradiol produces similar effects on the synthesis of ovalbumin, lysozyme and avidin. Biochim. Biophys. Acta, 145 (1967) 2o4-2o 7
207
PRELIMINARY NOTES TABLE I OVIDUCT NUCLEAR R N A IMMATURE CHICKS
POLYMERASE FOLLOWING DIETHYLSTILBGBTROL ADMINISTRATION TO
T h e a s s a y s y s t e m c o n t a i n e d o.o 5 m m o l e T r i s - m a l e a t e (pH 7.8), 0 . 2 5 / , m o l e u n l a b e l e d A T P , G T P , CTP, I.O/~C ? H ] U T P (2.3 C/mmole), 2.0/*moles MnC1 z a n d 2 . 0 / , m o l e s m e r c a p t o e t h a n o l . Nuclei were a d d e d in a m o u n t s e q u a l to i o - i o o / 2 g D N A . I n t h e h i g h ionic s t r e n g t h a s s a y s , a m m o n i u m s u l f a t e w a s a d d e d to 0.375 M. T h e final v o l u m e w a s i.o ml a n d i n c u b a t i o n s were carried o u t foi 15 m i n a t 38o followed b y a d d i t i o n of 7 % perchloric acid to a final c o n c e n t r a t i o n of 2 %. Carrier b o v i n e s e r u m a l b u m i n w a s a d d e d (0. 5 mg) a n d p r e c i p i t a t e s were w a s h e d t h r e e t i m e s in i % HC1Oa, t h e n w i t h 95 % ethanol, 0.02 M p o t a s s i u m acetate, p H 7.2. T h e final pellet was dissolved in 0. 5 ml NCS solubilizer (Nuclear Chicago) a n d cotinted in t o l u e n e - p h o s p h o r solution. R e s u l t s are e x p r e s s e d as #flmoles ? H ] U M P i n c o r p o r a t e d into acid-insoluble end p r o d u c t p e r m g D N A (nuclei) a d d e d to t h e i n c u b a t i o n . E a c h v a l u e r e p r e s e n t s t h e m e a n of 2 - 4 d e t e r m i n a t i o n s in at l e a s t two s e p a r a t e e x p e r i m e n t s .
Treatment
No h o r m o n e D i e t h y l s t i l b e s t r o l (24 h) D i e t h y l s t i l b e s t r o l (48 h)
?HI U M P /mg DNA (l~t~moles) Low ionic
High ionic
2.5 17.7 43.o
8. I 6o.3 99.7
Table I shows the change in oviduct nuclear RNA polymerase activity following a single 5-mg injection of diethylstilbestrol to the immature chick. The increase was not maximal 48 h after the hormone was given. The effect was similar both in the presence or absence of ammonium sulfate (0.375 M) in the polymerase assay in vitro.
The Endocrinology Branch, National Cancer Institute, National Institutes o~ Health, Belhesda, Md. (U.S.A.)
BERT W. O'MALLEY WILLIAM L. McGUIRE STANLEY G. KORENMAN
H. UI AND G. C. MUELLER, Proc. Natl. Acad. Sci. U.S., 5 ° (1963) 256. T. H. HAMILTON, Proc. Natl. Acad. Sci. U.S., 51 (1964) 83. J. GORSKI AND N. J . NELSON, Arch. Biochem. Biophys., i i o (1965) 284. K. L. BARKER, M. H. NIELSON AND J. C. WARREN, Endocrinology, 79 (1966) lO69. J. GORSKI, W. D. NOTEBOOM AND J. A. NICOLETTE, J. Cellular Comp. Physiol., 66 (1965) 91. J. GORSRI, J. Biol. Chem., 239 (1964) 889. J. W. BRANT AND A. V. NALBANDOV, Poultry Sci., 35 (1956) 692. R. HERTZ, R. M. FRAPS AND W. E. SEBRELL, Proc. Soc. Exptl. Biol. Med., 52 (1943) 142. S. G. KORENMAN AND B. W. O'MALLEY, Endocrinology, in t h e press. E. A. KABAT AND M. M. MAYER, Experimental Immunochemistry, T h o m a s , Springfield, Ili. 1961, p. 361. i i G. LITWAeK, Proc. Soc. Exptl. Biol. Med., 89 (1955) 4Ol. 12 S. G. KORENMAN AND B. W. O'MALLEY, Biochim. Biophys. Acta, 14o (1967) 174. 13 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951 ) 265. I 2 3 4 5 6 7 8 9 io
Received May 22nd, 1967 Biochim. Biophys. Acta, 145 (1967) 204-2o 7
PRELIMINARY NOTES
being 7o-S ribosomes '6. (d) By short-time labelling of RNA with a2Pl, and protein with EaH~lysine, a large percentage of each trichloroacetic acid-precipitable label was found in Peak II. Thus, functional polysomes appear to be associated with the membrane fraction of the cell in phage-infected as well as in normal E. coll. i am grateful to Dr. A. P. NYGAARD and Dr. J. G. HAUGE for support and helpful discussion of this work.
Department o/Biochemistry, University o/Bergen, Bergen (Norway) I 2 3 4 5 6 7 8 9 IO Ii 12 I3 14 15 16
LARS H A A R R
S. P. CHAMPE,Ann. Rev. Microbiol., 17 (1963) 87. M. SEKIGUCHI AND S. S. COHEN, J. Mol. Biol., 8 (1964) 638. 1R. B. KHESIN AND M. F. SHEMYAKIN, Biokhimiya, 27 (1962) 761. B. D. HALL, A. P. NYGAARD AND M. H. GREEN, J. Mol. Biol., 9 (1964) 143. K. F. BAUTZ, T. I~ASAI, E. REILI.Y AN]) F. i . BAUTZ, .Proc. Natl. Acad. Sci. U. S., 55 (1966) lO81. E. P. GEIDUSCHEK, L. SNYDER, A. J. E. COLVILL AND M. SARNAT, J. Mol. Biol., 19 (1966) 541 . J. C. SuiT, Biochirn. Biophys. dcta, 72 (1963) 488. D. SCttLESSlNGER, J. 3/Iol. Biol., 7 (1963) 569. P. A. HALLBERG AND J. G. HAUGE, Biochim. Biophys. Acta, 95 (1965) 80. L. D. MOORE AND W. W. UMBREIT, Biochim. Biophys. Acta, lO 3 (1965) 466. A. P. NYGAARD AND B. D. HALL, J. Mol. Biol., 9 (1964) 125. J. c. SUIT, J. Bacteriol., 84 (1962) lO61. A. P. NYGAARD AND B. D. HALL, Biochem. Biophys. Res. Commun., 42 (1963) 98. L. GROSSMAN, S. S. LEVlNE AND W . S. ALLISON, J. Mol. Biol., 3 (1961) 47. R. W. HENDLER AND J. TANI, Biochim. Biophys. Acta, 80 (1964) 294. G. MANGIAROTTI AND D. SCHLESSlNGER, J. Mol. Biol., 20 (1966) 123.
Received April i9th, 1967 Biochim. Biophys. Acta, 145 (I967) 2o2-2o4
BBA 91176
Estrogen stimulation of synthesis of specific activity in the immature chick oviduct
proteins and R N A polymerase
The precise mechanism of action of estrogen in the stimulation of growth of estrogen-sensitive tissues such as the uterus is unresolved, but there is good evidence for an early stimulation of RNA and protein synthesis 1-s. Estrogens have been reported to induce increased levels of m a n y enzymes in the uterus 4 and to stimulate incorporation of Iz4Cjglycine into protein, [aH~cytidine into RNA, and a~Pi, El*Cjacetate, and I14Clglucose into lipid, in this organ 5. GORSKIe has also demonstrated stimulation of rat uterus RNA polymerase after estrogen administration in vivo. Estrogens stimulate oviduct growth in newborn and older chicksL Formation of secretion granules within the oviduct glands and release of an albumin-like material into the lumen of the chick oviduct were reported to follow administration of estrogen and progesterone or estrogen and androgen combinationsL Avidin, the biotin-binding egg-white protein, has been noted to appear in the estrogen-stimulated chick oviduct Biochim. Biophys. Acta, 145 (1967) 2o4-2o7
PRELIMINARY NOTES
205
after a single dose of progesterone (HERTZ 8, KORENMAN AND O'MALLEy9). This communication deals with the changes in the oviduct proteins, ovalbumin and lysozyme, oviduct RNA polymerase activity, and the ability to synthesize avidin in response to progesterone after in vivo administration of diethylstilbestrol to immature chicks. Four-day-old female Rhode Island Red chicks were iniected subcutaneously with 5 mg diethylstilbestrol dissolved in sesame oil daily for 15 days. At the indicated times the animals were sacrificed. The oviducts were removed, weighed, homogenized, and an aliquot of the lO5 ooo ×g supernatant was analyzed for ovalbumin, lysozyme or avidin. Ova]bumin was precipitated b y specific antibody 1° and quantified b y protein estimation of the precipitate using the FOLIN--LowRY procedure 1°. Agar-gel diffusion studies on Ouchterlony plates demonstrated that the precipitating protein was identica] with ovalbumin. Lysozyme was assayed by the method of LITWACKn. Avidin was measured using an assay involving specific binding of the avidin to D-[carboxyl-14Clbiotin, adsorption of the 14C-labeled avidin-biotin complex onto bentonite, and trapping the complex on a Millipore filter 12. Measurements of the various proteins are expressed per g oviduct for convenience, as calculations on the basis of mg protein b y the method of LOWRY13 gave essentially the same results. Nuclear RNA polymerase was measured in vitro on nuclei isolated from oviducts at various times following a single dose of diethylstilbestrol. Oviducts were homogenized in buffer containing 0.32 M sucrose, o.ooi M MgC] 2, 0.0004 M KH2PO 4, 0.0004 M K2HPO 4, p H 6.7, and filtered through single-napp flannelette, washed with homogenizing buffer (three times) and centrifuged for 9 ° min at 5 ° ooo ×g in a solution containing 2.2 M sucrose, o.ooi M MgC12, 0.0035 M K2HPO4, 0.00o 7 M ATP, p H 6. 7. The nuclei were then resuspended in 0.05 M Tris-HC1, 30 % (v/v) glycerol, and o.ooi M inercaptoethanol, p H 7.6. The assay involves incorporation of labeled nucleotide into an acid-insoluble ribonuclease-sensitive product, and is dependent on exogenous nucleotides and is inhibited by deoxyribonuclease and actinomycin D. Details of the isolation method will be reported e]sewhere (W. L. McGUIRE AND B. W. O'MALLEY, unpublished observations). 3°°° I
DeS
]
I00-
8
IOOO
8 I
0
3
6
9
J2
J5 DAYS
18
21
24
27
0
~
e
9
12 r5 DAYS
18
2~
24
2Y
Fig. I. Effect of diethylstilbestrol (DES) a d m i n i s t r a t i o n on o v a l b u m i n levels in the i m m a t u r e chick oviduct. E a c h chick received 5 mg diethylstilbestrol in oil (subcutaneously) daily for 15 days. Chicks were sacrificed at the days indicated and o v a l b u m i n d e t e r m i n a t i o n s were carried o u t on the o v i d u c t s as described in the text. E a c h p o i n t r e p r e s e n t s the m e a n of 4 animals in a representative experiment. Fig. 2. Effect of diethylstilbestrol (DES) a d m i n i s t r a t i o n on lysozyme levels in the i m m a t u r e chick oviduct. Protocol is exactly as in Fig. I.
Biochim. Biophys. Acta, 145 (1967) 2o4-2o 7
206
PRELIMINARY NOTES
The effect of diethylstilbestrol administration on ovalbumin levels in the immature (unstimulated) chick oviduct is shown in Fig. I. The increase is minimal over the first 6 days. During this period morphological changes in cell differentiatien are taking place with appearance and proliferation of epithelial glands thought to be involved in ovalbumin synthesis (P. O. KOHLER AND B. W. O ' M A L L E Y , unpublished observations). Induction of ovalbumin synthesis is striking between 6 and 15 days. The level of ovalbumin at day 15 is 300 times the initial baseline concentration. When diethylstilbestrol is discontinued, a 62 °/o decrease in levels of this oviduct protein is observed over the next I I days. The fall probably represents a combination of decreased synthesis, degradation, and secretory loss into the gland lumen. A change in total oviduct mass occurs from 5 ~o mg in the immature chick to 1. 5 to 3 g after 3 weeks of estrogen treatment. We know of no other tissue capable of such a marked growth response to a hormone. A similar induction of oviduct lysozyme synthesis occurs over the same period of diethylstilbestrol treatment (Fig. 2). Withdrawal of the hormone again leads to a 65 % fall in specific activity of the enzyme over the next I I days. Indaetion of avidin synthesis in the chick oviduct after administration of progesterone in vivo will be presented in detail elsewhere 9. However, we have examined the role that diethylstilbestrol administration plays in the ability of the oviduct to respond to a progestational stimulus (Fig. 3). Progesterone-induced avidin synthesis is low in the hor-
25
2o
0
I0 OAYS OFOES(5mg)
t5
Fig. 3- E f f e c t of d i e t h y l s t i l b e s t r o l (DES) a d m i n i s t r a t i o n on t h e chick o v i d u c t c a p a c i t y to s y n t h e s i z e avidin. D i e t h y l s t i l b e s t r o l w a s a d m i n i s t e r e d as in Fig. I. A single 5-rag injection of p r o g e s t e r o n e in p r o p y l e n e glycol ( s u b c u t a n e o u s l y ) w a s a d m i n i s t e r e d 24 h prior to sacrificing t h e a n i m a l s . Avidin ( m e a n of io a n i m a l s w i t h range) c o n t e n t w a s m e a s u r e d in each o v i d u c t as described in t h e t e x t a n d is r e p r e s e n t e d b y t h e b a r g r a p h . T h e e x p e r i m e n t w a s r e p e a t e d twice. No m e a s u r a b l e a v i d i n is p r e s e n t in o v i d u c t s of d i e t h y l s t i l b e s t r o l - t r e a t e d chicks w h i c h h a v e n o t received progesterone.
monally immature oviduct (day o) but the capacity for avidin synthesis increases a great deal by d a y 6 of diethylstilbestrol administration. Further estrogen reduces the capacity of the oviduct to synthesize avidin. If the diethylstilbestrol treatment is extended beyond 21 days, only 1.5-4.o #g avidin/g oviduct is found after induction by progesterone. Estradiol produces similar effects on the synthesis of ovalbumin, lysozyme and avidin. Biochim. Biophys. Acta, 145 (1967) 2o4-2o 7
207
PRELIMINARY NOTES TABLE I OVIDUCT NUCLEAR R N A IMMATURE CHICKS
POLYMERASE FOLLOWING DIETHYLSTILBGBTROL ADMINISTRATION TO
T h e a s s a y s y s t e m c o n t a i n e d o.o 5 m m o l e T r i s - m a l e a t e (pH 7.8), 0 . 2 5 / , m o l e u n l a b e l e d A T P , G T P , CTP, I.O/~C ? H ] U T P (2.3 C/mmole), 2.0/*moles MnC1 z a n d 2 . 0 / , m o l e s m e r c a p t o e t h a n o l . Nuclei were a d d e d in a m o u n t s e q u a l to i o - i o o / 2 g D N A . I n t h e h i g h ionic s t r e n g t h a s s a y s , a m m o n i u m s u l f a t e w a s a d d e d to 0.375 M. T h e final v o l u m e w a s i.o ml a n d i n c u b a t i o n s were carried o u t foi 15 m i n a t 38o followed b y a d d i t i o n of 7 % perchloric acid to a final c o n c e n t r a t i o n of 2 %. Carrier b o v i n e s e r u m a l b u m i n w a s a d d e d (0. 5 mg) a n d p r e c i p i t a t e s were w a s h e d t h r e e t i m e s in i % HC1Oa, t h e n w i t h 95 % ethanol, 0.02 M p o t a s s i u m acetate, p H 7.2. T h e final pellet was dissolved in 0. 5 ml NCS solubilizer (Nuclear Chicago) a n d cotinted in t o l u e n e - p h o s p h o r solution. R e s u l t s are e x p r e s s e d as #flmoles ? H ] U M P i n c o r p o r a t e d into acid-insoluble end p r o d u c t p e r m g D N A (nuclei) a d d e d to t h e i n c u b a t i o n . E a c h v a l u e r e p r e s e n t s t h e m e a n of 2 - 4 d e t e r m i n a t i o n s in at l e a s t two s e p a r a t e e x p e r i m e n t s .
Treatment
No h o r m o n e D i e t h y l s t i l b e s t r o l (24 h) D i e t h y l s t i l b e s t r o l (48 h)
?HI U M P /mg DNA (l~t~moles) Low ionic
High ionic
2.5 17.7 43.o
8. I 6o.3 99.7
Table I shows the change in oviduct nuclear RNA polymerase activity following a single 5-mg injection of diethylstilbestrol to the immature chick. The increase was not maximal 48 h after the hormone was given. The effect was similar both in the presence or absence of ammonium sulfate (0.375 M) in the polymerase assay in vitro.
The Endocrinology Branch, National Cancer Institute, National Institutes o~ Health, Belhesda, Md. (U.S.A.)
BERT W. O'MALLEY WILLIAM L. McGUIRE STANLEY G. KORENMAN
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Received May 22nd, 1967 Biochim. Biophys. Acta, 145 (1967) 204-2o 7