lation of phosvitin synthesis by estradiol are characterized by a lag of 12 and 6 hours, respectively. If during these lag phases RNA synthesis is blocked by a- ...
THE Jovmn~ OF I(IOLOGICAL CHEMISTRY Vol. 248, No. 15, Issue uf August 10, PP. 5262-5266, Prznted in C.S.A.
1073
Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during Phosvitin Induction by 17P-Estradiol in Inuuature Chicks (Received for publication,
January
18, 1973)
JEAN-PIERRE .JOST, ROSNARIE KELLER, AND CHRISTA DIERKS-VENTLING From
the Fyieclrich
Miescher
Institut,
Base& Switxedancl
SUMMARY
l~:Xl?I~;RIME~TAL
Phosvitin is one of the main proteins of egg yolk (l-5). Its production by the laying hen during vitellogenesis is mediated by estradiol (3, 5), yet phosvitin can also be induced in roosters by injection of a large dose of estradiol (6-8). Phosvitin is synthesized in the liver (4, 9, 10) from where it is secreted into the blood. After a single injection of estradiol into a rooster, the appearance of phosvitin in the plasma is delayed by 20 hours (primary stimulation) (7, 8, 11). A second injection of estradiol given 10 to 14 days after the first one, at a time when the phosvitin concentration in the plasma has returned to the base-line of the non-cstrogenized controls, produces within 6 hours a sharp increase of phosvitin synthesis (7, 11). Thus, the secondary stimulation is characterized by a much shorter lag period. These observations demonstrate that the estradiol-induced synthesis of phosvitin represents a good model to study the molecular events of hormone action. The question of whether DNA or RNA synthesis, or both, arc absolutely required for phosvitin induction has been dealt with by several groups in the past years. Thus, Jailkhani and Talwar (12) recently reported that in immature estrogenized chicks inhibition of DNA synthesis resulted in the failure of the target organ to produce phosvitin. The aut.hors used mitomycin C and hydroxyurea among
PROCIIDURE
dlaterials
[5-zH]Uridine (specific activity 3 Ci per mmole) and [2-1aC’]thymidine (specific activity 50 mCi per mmole) were purchased from Radiochemical Centre, Amersham, Euglaud. 17P-Estradiol was obtained from CIBA-GEIGY Limited, Uascl, Switzerland. Professor T. Wicland of the Max Planck Institute, Heidelberg, Germany, generously supplied the a-amanitin. Hydroxyurea n-as purchased from Fluka, Fuchs, Switzerland. Methods
white leghorn chicks were supplied locally, maintained in electrically heated brooders, and were fed a standard diet ad lib&m. Estradiol was dissolved in propylene glycol (10 to 30 mg per ml). A constant volume of 0.2 ml of hormone solution/100 g body weight was injected intramuscularly into the leg. The primary stimulatiou is defined as the results obtained after the first injection of estradiol. The secondary stimulation by the steroid was usually initiated 10 to 14 days after the first injection. The controls received propylene glycol only. Since in immature chicks no sex difference was observed for the stimulation of phosvitin synthesis, we used a mixed population of male and female birds. Hydroxyurea and
5262
Treatment
of Animals-Newborn
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1. During the first 48 hours following estrogenization a total increase in liver weight, DNA, RNA, and liver protein is observed. 2. Inhibition of DNA synthesis with 300 mg of hydroxyurea/l00 g body weight (injected every 12 hours) does not impair the primary or secondary stimulation of phosvitin synthesis. 3. The early kinetics of the primary and secondary stimulation of phosvitin synthesis by estradiol are characterized by a lag of 12 and 6 hours, respectively. If during these lag phases RNA synthesis is blocked by a-amanitin (0.02 mg per kg body weight, injected twice in 12 hours interval), no further synthesis of phosvitin is observed. 4. Total polysomal mRNA synthesis doubles during the oc-amanitin-sensitive period after the primary and secondary stimulation. During that same period, but after the secondary stimulation only, rRNA synthesis increases by about 90%.
other compounds to inhibit DNB synthesis. Greeiigard et al. (8) studied the primary stimulation and injected actinomyciu D into estrogenized roosters with the result that phosvitiu synthesis was abolished, and they concluded that RNA synthesis was a prerequisite for phosvitin induction. In contrast, Beuving and Gruber (7) who used the same drug to study RNA requirements in roosters during the secondary stimulation discovered that 110 inhibition of phosvitin synthesis took place. We reinvestigated the requirements of DNA aud RNA\ synthesis during both the primary and secondary stimulatiou of phosvitin synthesis using as inhibitors hydroxyurea and cr-amauitiu, respectively. We chose immature chicks rather thau roosters because in the former sex hormones in general can be excluded. In this communication we show that DNA synthesis is not required for either primary or secondary stimulation, but that RNA synthesis is absolutely necessary during both periods. Also investigated was the total polysomal mRNA synthesis which was found to be siguificantly increased during the 01amanitin-sensitive phases of the primary and secondary stimulation, while the rRNA synthesis was only enhanced during the secondary stimulation.
5263 a-amanitin were dissolved in 0.85y0 sodium chloride solution and administered intraperitoneally in amounts indicated in the text. Determination of Phosvitin in Plasma-Blood was collected in conical tubes containing 50 units of heparin per ml of blood and the plasma \vas obtained by centrifugation at 2000 x g for 10 min. Total plasma phosphoproteins were extracted by calcium precipitation accordin g to Heald and McLachlan (2) and phosvitin was isolated on DEAE-cellulose as described by Connelly and ‘Taborsky (13). For routine determinations of phosvitin the method by Beuving and Gruber (7) was used. Determination of Total DNA, RNA, and Protein in Livers-An aliquot (0.1 ml) of a 10% liver homogenate (w/v) in water was precipitated with 5 ml of 10% trichloroacetic acid. The precipitate was extracted consecutively with 2 ml of cold acetone, twice with 2 ml of cold ethanol-ether-chloroform (2:2: 1, v/v/v) and once with 2 ml of cold acetone. DNA was determined in the precipitate according to Burton (14), RNA according to Fleck and Munro (15), and proteins by means of the biuret reaction
(16).
RESULTS
Synthesis of Phosvitin, Total Liver DNA, RNA, and Proteins during Primary and Secondary Stimulation by &tradiol-Fig. 1 shows that under our experimental conditions the primary stimulation (Curve B) was characterized by a lag of about 12 hours prior to a gradual increase in phosvitin synthesis, whereas
primary
and
secondary
stimulation
of phosvitin
synthesis
by
estradiol led us to investigate whether DNA synthesis was required for the induction of phosvitin. As a specific inhibitor of DNA synthesis we chose hydroxyurea which is known to block DNA synthesis in animal tissues by inhibiting nucleotide reductase (20, 21). A dose response curve for the inhibition of DNA synthesis by hydroxyurea was established as follows. Chicks (100 g body weight) were injected intraperitoneally with 100, 200, 300, and 400 mg of hydroxyurea, respectively. Four hours later,
groups
of three
chicks
each
were
labeled
for
50 min
with
2 PC1 of [Wlthymidine. The thymidine incorporation was determined in the 10% trichloroacetic acid precipitate of the liver homogenate (10% in water) and the incorporation was
. -: . 1 A,
fi 10
20
30 40 5 IO HOURS FIG. 1. Early kinetics of the primary and secondary stimulation of phosvitin synthesis by estradiol. Groups of three to four chicks each (150 g + 15 body weight) received a single injection of 50 mg of estradiol per kg. Ten days prior to the secondary stimulation, chicks (60 g body weight) received a first injection of 50 mg of estradiol per kg. At the time indicated, chicks were killed and alkali-labile phosphorus and total plasma proteins were deter-
6
mined in the plasma. primary, sis by
A,A’
represent
the controls,
B,B’
the
and C,C’ the secondary stimulation of phosvitin syntheestradiol. Each point represents the mean f S.E.M. of
three to four chicks.
0
10
20
30
40
1
0 HC NJI PS
10
20
30
40
I
FIG. 2. Total liver weight, DNA, RNA, and protein content of livers from estrogenized chicks. This experiment is part of that described in Fig. 4. A represents the controls, B the primary, and C the secondary stimulation of phosvitin synthesis by estradiol.
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Isolation of Polysomes and mRNA-Total polysomes of livers were prepared according to Blobel and Potter (17), except that we purified the polysomes through a discontinuous sucrose gradient as described by Lee et al. (18). RNA was extracted from the polysomcs with cold phenol, and poly(A)-rich mRNA was isolated by means of cellulose nitrate Millipore filters (19).
the secondary stimulation (Curve C) already displayed a sharp increase in phosvitin synthesis 6 hours after estradiol injection (the identity of plasma phosphoproteins with egg yolk was demonstrated by column chromatography on DEAE-cellulose). In the nonestrogen&d controls (Curve A) no trace of phosvitin could be detected in the plasma at any time. Over a period of 48 hours the plasma proteins of the estrogenized birds increased 1.8 and 2.5.fold for the primary and the secondary stimulation, respectively (Fig. 1, B’ and C’). Estradiol injections into immature chicks led to remarkable changes of actual liver weight, DNA, RNA, and protein content of the livers (Fig. 2). During both the primary and secondary stimulation by estradiol the a-et weight of the livers and the protein content increased 1.6.fold over the controls (during the same period the total body weight of estrogenized birds did not increase over that of the controls). Total liver RNA increased 1.6-fold during both the primary and secondary stimulation, albeit a delay of 6 hours was observed for the primary stimulation. The total amount of DNA increased to a maximum of about 1.3.fold. EJect of Inhibition of DNA Synthesis on Induction of Phosvitin by E&radio&l’he observed increase in liver DNz4 during the
Effecl
of
hyd?oxyu?'ea of phosvitin
on primary synthesis
and secondary by 1’7p-estradiol
stimulation
All chicks (150 f 15 g body weight), except the controls, received 50 mg of estradiol per kg. Gro\lps of four to six chicks each received hydroxyurea (300 mg/lOO g body weight) at the time indicated in the table and 12 hours later. All chicks were killed 24 hours (Experiment A) and 48 hours (Experiment B) after estrogenization, and alkali-labile phosphorus was determined in the plasma. Bach value represents the mean f S.E.M. of four to six chicks. The number of animals is in parentheses. Primary
stimulation
Secondary
stimulation
F‘ercent
Experiment
age of inhibiton
Pi
Percentage of inhibition
Pi
/q/ml
plasma
A
I
1
100 200 300 mg HYDROXYUREA/lOOg
4 I
400
BODY WEIGHT
FIG. 3. Dose-response curve of the inhibition of DNA synthesis by hydroxpurea. The experiments were designed as described in the text. The insetrepresents the time course of the inhibition of DNA and R,NA synthesis in the livers of 100-g chicks receiving a single injection of 300 mg of hydroxyurea. Each point represents the mean i S.E.M. of three chicks. corrected for the trichloroacetic acid-soluble counts. With 300 to 400 mg of hydrosyurea per 100 g body weight, DNA synthesis could be inhibited by about 97% (Fig. 3). The inset of Fig. 3 shows the time course of the inhibition of DNA and RNA synthesis in the liver of chicks receiving a single injection of 300 mg of hydroryurea/lOO g body weight. At the times indicated in the figure, three chicks were labeled for 50 min with 1.6 &i of [‘4C]thymidine and 30 PCi of [WI]uridine. Again the incorporation of the labeled nucleosidcs was determined in the trichloroacetic acid precipitate as described above. It can be clearly seen that during the first 12 hours while DNA was inhibited by about 90 to 95c/, RNA was only inhibited by 33%. Although a partial RX-1 iilhibition could not be circumvented, hydroxyurea is still to be preferred to mitomycin C, which under similar conditions inhibited RNh synthesis by over 507,’ (see also References 22 to 24). Next we tested the effect of hydroxyurea on the induction of phosvitin by estradiol (Table I). Groups of four to six chicks each received a siugle injection of 50 mg of estradiol per kg and hydroxyurea (300 mg/lOO g) was injected at the times indicated and 12 hours later (Experiment A). All birds were killed 24 hours after estradiol treatment and the phosvitin was determined in the plasma. At no time could any inhibition of phosvitin synthesis be observed during the primary stimulation, and even for the secondary stimulation inhibition was minimal. In Experiment B we investigated whether an effect of hydroxyurea would be apparent at a time later than 24 hours. For this purpose groups of four to six chicks each were injected simul1 J. P. Jost, unpublished
observation.
0
311 7.5 f 5.7 7.1 5.8 4.0 0.2
f f f f f
6)
2 (6)
2 2 7 2 4
(4) (4) (4) (4) (4)
10 2.3 0 0 0
23.4 35.1 28.7 26.4 25.G
f f III f zt
2 5 6 2 2
(4) 24.5 (4) 0 (4) 7.4 (4) 14.8 (4) 17.4
B Non-estrogenized chicks. 0 (6) Estrogenized chick: 33 5.2 f G (G) Estrogenized chick! 3 receiving hydroxyurea at 5'0.0 i 7 (0) zero time..
05.0 11
0 (6) f 8 (6) 0
116.4 f
4 (6)/
0
taneously with 50 mg of estradiol per kg and 300 mg of hydroxyurea/100 g body weight. Injections of hydroxyurea were given three more times at 12hour intervals, and the birds were killed at 48 hours. Again the induction of phosvitin was unimpaired. Thus, the estrogen-stimulated synthesis of phosvitin was unaffected under conditions where there was 95% inhibition of DNA4 synthesis. EJect of Inhibition of RNA Synthesis on Induction of Phosvilin by Estradiol-In Fig. 2 we showed that the total RSA content of the liver of estrogenized birds increased 1.6.fold during the primary and secondary stimulation of phosvitin synthesis. In an attempt to determine whether RNA synthesis was required for the induction of phosvitin synthesis, experiments using acamanitin, a specific inhibitor of nucleoplasmic DXA-dependent RNA polymerase (25, 26), were carried out. Fig. 4 shows a dose-response curve of cY-amanitin for the inhibition of total RNA synthesis in the liver of immature chicks. The experiment was designed as described for hydroxyurea. A maximum of RNA inhibition was observed with a single dose of 0.05 mg of QIamanitin per kg. Intraperitoneal injections of 0.1 mg of aamanitin per kg or more were lethal within 24 hours. For a 1OO70 survival over a period of 48 hours, the total dose of cramanitin should not exceed 0.05 mg per kg. Inhibition of total RNA synthesis by a single injection of 0.02 mg of Lu-amanitin per kg was studied as a function of time (inset of Fig. 4). The
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I
Non-estrogenized chicks, Estrogenizedchickz Estrogenized chick5 receiving hydroxyurea at time : -4 hours. Zero time. +4 hours.. +8 hours. +12 horns.
5265 TABLE
II
primary synthesis
and
100
Effect
of wamanitin
on phosvilin
ao-
-
\\ 20
0
4
a 12 HOURS
Primary
‘5
stimulation
Secondary
stimulation
ercent-
I Pi
pg/ml
I
,
1
0.1
0 BODY
WEIGHT
4. Dose-response curve of the inhibition of RNA synthesis by a-amanitin. The experimental design is described in the text. The inset represents the time course of the inhibition of RNA synthesis in the livers of 100-g chicks receiving a single injection of 0.02 mg of a-amanitin. Each poi?zt represents the mean f S.E.M. of three chicks. FIG.
experimental design was as described above for hydroxyurea. It call be seen that ar-amanitin drastically decreased the rate of total RNA synthesis for up to 12 hours. Next me tested the effect of 0 02 mg of ac-amanitin per kg on the induction of phosvitin during the primary and secondary stimulation by estradiol. Groups of four to six chicks each (150 g) received a single dose of 50 mg of estradiol per kg, and oc-amanitin (0.02 mg per kg) lvas injected at the time indicated and 12 hours later. All birds were killed 30 hours after estradiol treatment and the phosvitin was determined in the plasma. ai-8manitin inhibited both the primary and the secondary stimulation of phosvitin synthesis by estradiol (Table II). During the primary stimulation the drug completely inhibited phosvitin induction if injected less than 14 hours after estrogcnization. The secondary stimulation was totally abolished by the drug only during the first 4 hours following injection of the hormone. Polysomal Secondary
RNA Xlimulation
Synthesis during of Phosvitin
Early Phase of Primary Synthesis by Estradiol--We
and
demonstrated above that estradiol increased the total RNA synthesis in the livers of estrogenized chicks. This fact and the observation that cu-amanitin inhibited the early phase of both the primary and secondary stimulation by estradiol suggested that new mRNA and rRNA are synthesized. In the following experiment 1T-e labeled groups of three estrogenizcd chicks for 2 hours with 200 &Yi of [“H]uridine/lOO g. Two time points were chosen: 0 to 2 hours and 4 to 6 hours following the injection of the steroid. Total polysomes were prepared frorn the livers; mRNA and rRNS were isolated as described under “Experimental Procedure.” Table III shows that during the primary stimulation the rate of mRNX increased to 100% and more, Tvhereas rRNh synthesis did not increase at the t’wo time points tested (an increase in rRNA synthesis occurs only later). For the secondary stimulation, however, the rates of synthesis of both mRKA and rRNA were found to be significantly increased 2 hours after estradiol t,restmcnt.
Non-estrogenized chicks. Estrogenized chick s!l8.5 Estrogenized chick S receiving Namanitin at time : -4 hours. 0.1 Zero time. +4 hours.. +lOhours...... +14 hours. _. 2.5 +18 hours.. 8.4
age of inhibition
pi
plasma
pg/ml
pzes11ie
0 :
2
I:;
0
i 0.2 (4) 93 0 (4) 100 0 (4) 100 0 (4) 100 f f
0.G 0.5
(4) (4)
68.0
i
6)
7
0 1.2
i
1.0 f 48.8
2
(G)
0
(41
100 98 98.5
(4)
1 (4)
zk 5
(4)
28.3
88 54
DISCUSSION
Although the increase in DNA content of the liver of estrogenized birds precedes the appearance of phosvitin synthesis, there is no evidence that the two phenomena are related, since under our experimental conditions hgdroxyurea (300 rng/lOO g injected every 12 hours) had 110 inhibitory effect, 011 the formation of phosvitin. It is thus unlikely that the lag betmeen the primary and the secondary stimulation are due to an increase in the number of target cells. In sharp contrast to our findings, Jailkhani and Talwar (12) reported that in imrnature chicks injections of only 8 mg of hydroxyurea/lOO g given every 24 hours completely suppressed phosvitin induction. The reason for these differences remains unresolved. That RNA synthesis is required for the stimulation of phosvitin induction has been shown through inhibition by actinomycin D (5, 7, 8). This compound was shown to inhibit the primary but not the secondary stimulation (7), which led to the rather premature conclusion that during the secondary stimulation esbradiol controls phosvitin synthesis on a translational level (7). Using a-amanitin as inhibitor of RNA polymerase (25, 26)) we found that the drug completely suppressed phosvitin induction if given within the first 10 hours following the primary stimulation or within 4 hours after the secondary stimulation. This finding correlates well with the observed differences in lag of phosvitin synthesis between the primary and secondary stimulation. The discrepancy between the results of Beuving and Gruber (7) and ours could possibly be explained by their initiation of the secondary stimulation at a time when the birds were still synthesizing phosvitin as a result of the primary stimulation (7).
In recent years, much knowledge about the species of RNA induced by estradiol has been gained (27, 28). The most im-
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0.01 0.05 mg d;AMANITIN/kg
of
All chicks (150 i 15 g body weight) except the controls received 50 mg of estradiol per kg. Groups of four to six chicks each received cr-amanitin (0.02 mg per kg body weight) at the time indicated in t,he table and 12 hours after estrogcnization. All chicks were killed 30 hours after estrogenization. $lkali-labile phosphorus was determined in the plasma. Each value represents the mean + S.E.M. of four to six chicks. The number of animals is in parentheses.
6 f600’ ;400 x
stimulation
secondary
by estradiol
5266 TABLE Effect
of e&radio1
on rate
of polysomal
RNA
synthesis
during
III primary
and secondary
stimulation
of phosvitin
synthesis
All chicks (150 f 15 g body weight), except the controls, received 50 mg of estradiol per kg body weight. Groups of three chicks each were pulse-labeled for 2 hours with 300 pCi of [3H]uridine and RNA was isolated from the liver polysomes as described under “Experimental Procedure.” Each value represents the mean f S.E.M. of three determinations. 04
2
hours
4 4
6
hours
Stimulation Polysomal mRNA
Polysomal rRNA
% Primary Controls Estrogenixed Secondary Controls. Estrogenized
%
dpm.lOP/mg
chicks.
132.7 348.5
f f
45 10
162
24.6 26.0
f 0.3 zt 0.4
chicks..
103.9 249.5
f Ik
12.9 14.3
140
46.3 70.7
f f
1.7 3.1
dcknowLedgments-We should like to thank Drs. H. Bloch, J. Gordon, D. Monard, and H. I’. Schnebli for valuable criticism and discussion. REFERENCES 1. MECHAM, 2. 3. 4. 5.
D. K., AND OLCOTT, H. S. (1949) J. Amer. Chem. Sot. 71, 3670-3679 HEALD, P. J., AND MCLACHLAN, P. M. (1963) Biochem. J. 87, 57 l-576 HEALD, P. J., AND MCLACHLAN, P. M. (1964) Biochem. J. 92, 51-55 RUDACIC, D., AND WALLACE, R. A. (1968) Biochim. Biophys. Acta 166, 299-301 WITTLIFF, J. L., AND KENNEY, F. T. (1972) Biochim. Biophys. Acta 269, 485-492
IIlCXaSe
dpm. IO-=/mg
%
Polysomal rRNA
InCreaSe %
dpm.lo-~/mg
5
360.0 721.3
f f
15.4 45.4
100
40.6 26.4
f f
1.1 4.7
0
52
194.8 720.2
f f
19.9 20.5
269
29.4 57.3
f f
0.6 0.1
94
6. MCDONALD, M. R., AND RIDDLE, 0. (1945) J. Biol. Chem. 169 445-464 7. BEUVING, G., AND GRUBER, M. (1971) Biochim. Biophys. Acta 233, 529-536 8. GBEENGUD, O., GORDON, M., SMITH, M. A., AND Acs, G. (1964) J. Biol. Chem. 239, 2079-2082 9. GREENGARD, O., SENTENAC, A., AND Acs, G. (1965) J. Biol. Chem. 240, 1687-1691 10. HEALD, P. J., AND MACLACHLAN, P. M. (1965) Biochem. J. 94, 32-39 11. JAILKHANI, B. L., BND TALWAR, G. P. (1972) Nature New Biol. 236, 239-240 12. JAILI~HANI, B. L., AND TALWAR, G. P. (1972) Nature New Biol. 239, 240-241 13. CONNELLY, C., AND TABORSKY, G. (1961) J. Biol. Chem. 236, 1364-1368 14. BURTON, K. (1956) Biochem. J. 62, 315-323 15. FLECK, A., AND MUNRO, H. N. (1962) Biochim. Biophys. Acta 66, 571-583 16. LAYNE, E. (1957) Methods Enzymol. 3, 447-454 17. BLOBEL, G.. AND POTTER, V. R. (1967) J. Mol. Biol. 26, 279292; 263-361 18. LEE. S. Y.. KRSM.QNOVIC. V.. AND BRAWERMAN, G. (1971) Biochemislry 10, 895-900’ ’ 19. BRA4WERMAN, G., MANDECKI, J., END LEE, S. Y. (1972) Biochemistry 11, 637-641 20. SCHWARTZ, H. S., GAROFaLO, M., STERBERG, S. S., AND PHILIPS, F. S. (1965) Cancer Res. 26, 1867-1870 21. YOUNG, C. W., SCHOCHETMAN, G., AND KARNOFSKY, D. A. (1967) Cancer Res. 27, 526-533 22. PRICER, W. E., JR., AND WEISSB~CH, A. (1965) Biochemistry 4, 200-206 23. LIPSETT. M. N.. AND WEISSBACH, A. (1965) Biochemistry 4, 206-2li 24. KERST~;N, H., KERSTEN, W., LEOPOLD, G., AND SCHNIEDERS, B. (1964) Biochim. Biophys. Acta 80, 521-523 25. WIEL.~ND,'T. (1972) Nat&u&senschajt 69, 225-231 26. TAT.\, J. R., HAMILTON, M. J., AND SHIELDS, D. (1972)Nature New Biol. 238, 161-164 27. TATA, J. R. (1966) Progr. Nucl. Acid Res. Mol. Biol. 6, 191-250 28. HAMILTON, T. H. (1971) in The Biochemistry of Steroid Hormone Action, (SMELLIE, R. M. S., ed) p. 49, Academic Press,
New York 29. HAHN,W. E., SCHJEIDE, Nat. Acad. Sci. 30. MXENPBX, P. H., 8, 4926-4935 31. M~ENPU, P. H. 971-974
0. A., AND GORBMAN, A. (1969) Proc. U. S. A. 62, 112-119 AND BERNFIELD, M. R. (1969) Biochemistry (1972)
Biochem.
Biophys.
Res. Commun.
47,
Downloaded from www.jbc.org by guest, on July 21, 2011
portant information with regard to the avian system came from two groups of investigators. Hahn et al. (29) showed through DNA-RNA hybridization studies that the RNA molecules induced by estradiol in the liver and the oviduct were not homologous and that species of RNA other than ribosomal RNA were synthesized. At the same time ?JIBenp&& and Bernfield (30, 31) found that estradiol increased two types of seryl-tRNA in the liver of estrogenized roosters. Having given evidence above that RNA synthesis is required during the lag period of phosvitin induction, we proceeded to study the rate of total polysomal RNA synthesis during the cr-amanitin-sensitive phases of the primary and secondary stimulation of phosvitin synthesis and found that the rate of total polysomal mRNA increased by 100 y. or more in both cases. In contrast, the rate of rRNA synthesis did not increase during the first 6 hours of the primary stimulat,ion (an increase in rRNA synthesis occurs only later), whereas a significant increase in rRNA synthesis was observed for the secondary stimulation. These findings may partially explain the time difference in lag phases of the primary and secondary stimulation. At this point, however, it is not possible to say how much of the newly synthesized mRNA is phosvitin mRNB. Any conclusion about the role of transcriptional and translational control of the primary and secondary stimulation of phosvitin synthesis can only be drawn after the exact rate of phosvitin mRNS synthesis has been determined.
Polysomal mRNA
