Proc. Natl. Acad. Sci. USA Vol. 76, No. 3, pp. 1049-1053, March 1979
Biochemistry
Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene* (nuclear transcription/cloned DNA/hormone action/intervening gene sequences/precursor RNA)
GEORGE E. SWANECK, JEFFREY L. NORDSTROM, FRITZ KREUZALER, MING-JER TSAI, AND BERT W. O'MALLEY Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
Communicated by Sidney P. Colowick, November 27, 1978
The transcription of structural and intervening ABSTRACT se uences of the chicken ovalbumin gene was studied in nuclei isoated from the oviduct, liver, and spleen of chickens in different states of estrogen stimulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [3HJRNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [3H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription. Previous studies on the regulation of gene expression in eukaryotes have shown that steroid hormones induce synthesis of specific proteins in vivo by increasing the concentration of their mRNAs (1-3). Injections of estrogen into chickens that had been prestimulated with estrogen and then withdrawn from all hormone (secondary stimulation) induced the accumulation of ovalbumin mRNA (mRNAOv) (4-7). Recently, it has been shown that the structural sequences of the ovalbumin gene (8-10) and globin gene are interrupted by multiple regions of nonstructural intervening sequences. In addition, the 15S precursor to globin mRNA was shown to contain both structural and intervening sequences of the globin gene (11). Similarly, transcripts of both types of ovalbumin sequences accumulated in vivo in oviduct nuclei after stimulation with estrogen (12). In addition, we have demonstrated the existence of high-mo-
lecular-weight species of ovalbumin RNA in nuclear extracts of oviducts stimulated chronically with estrogen (12). Because these molecules contain sequences complementary to intervening as well as to structural sequences of the ovalbumin gene, it is likely that these RNAs are precursors to mature
mRNA0v.
In an attempt to define the mechanism of steroid hormoneinduced accumulation of mRNA0v, we have studied the rate of transcription of the ovalbumin gene in vitro and the size of the ovalbumin gene transcripts in vivo. In the present study, we report that secondary stimulation of chickens with diethylstilbestrol (DES) elevates the extent of transcription of both structural and intervening sequences of the ovalbumin gene as assayed in isolated oviduct nuclei. We also demonstrate that DES stimulation increases the level of high-molecular-weight ovalbumin sequence-containing RNA in vivo in addition to that of mature mRNA0v. These results further support the hypothesis that an increase in the rate of transcription of the ovalbumin gene appears to be the major mechanism by which estrogen elevates the intracellular level of mRNA., (4, 5). MATERIALS AND METHODS Hormone Treatment. Estrogen-stimulated oviducts were obtained from White Leghorn chickens that were implanted weekly with a 20-mg pellet of DES. This pellet provided continuous release of DES for 8-9 days. Estrogen-withdrawn oviducts were obtained from chickens that had received daily subcutaneous injections of 2.5 mg of DES for 14 days and were subsequently withdrawn from hormone for periods up to 14 days. For experiments involving acute stimulation with estrogen, one injection of 2.5 mg of DES was given to hormonewithdrawn chickens. Oviducts were collected at the time intervals indicated in the text. Synthesis and Isolation of [3HIRNA from Nuclei. Nuclei were purified from oviducts of either hormone-stimulated or withdrawn chickens as described (12). [3H]RNA was transcribed in isolated nuclei in a reaction mixture of 5 ml that contained 0.8 mM each of ATP, GTP, and UTP, 0.014 mM [3H]CTP (35.4 Ci/mmol, 1 Ci = 3.70 X 1010 Bq), 3 mM MgCI2, 2.5 mM MnCI2, 3 mM dithiothreitol, 0.5 mM EDTA, 100 mM KCI, 12.5% (vol/vol) glycerol, 50 mM Tris-HCl (pH 7.8), and 0.8-2.2 X 108 nuclei per ml. After 30 min at 37°C, the [3H]RNA synthesized was isolated by the procedure of Roop et al. (12). Abbreviations: DES, diethylstilbestrol; pOV230 DNA, plasmid DNA containing a nearly full-length ovalbumin cDNA insert (13); pOVl.8 DNA, plasmid DNA containing the 1.8-kilobase EcoRI fragment of the natural ovalbumin gene (14); pOV2.4 DNA, plasmid DNA containing the 2.4-kilobase EcoRI fragment of the natural ovalbumin gene (14); mRNAov, ovalbumin mRNA. * This is paper no. 22 in a series of publications from our laboratory dealing with hormone regulation of gene expression in the chicken oviduct. Paper no. 21 is ref. 15.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1049
1050
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Swaneck et al.
Hybridization of [3HJRNA to DNA Filters. Filters containing pOV230, pOV1.8, or pOV2.4 DNA were prepared as described (9, 12-14). All preparations of cloned DNA were performed in a P3 facility and all procedures were carried out in accordance with National Institutes of Health guidelines. Ovalbumin structural and intervening sequence transcripts were measured by hybridizing the in vitro [3H]RNA to nitrocellulose filters containing pOV230 or pOV2.4 or pOVl.8 DNA as described (12). Briefly, 1-4 X 106 cpm of [3H]RNA was hybridized to DNA filters (1.2 ,ug) at 420C for 18 hr in 50% (vol/ vol) formamide/0.21 M NaCl/0.021 M Na citrate at pH 7.0 in the presence and absence of competitor (7.5 ,tg of unlabeled mRNAOv or 9 ,ug of unlabeled cRNA transcribed from pOV2.4 or pOVl.8 DNA). At the end of incubation, the filters were extensively washed in 0.30 M NaCl/0.030 M Na citrate at pH 7 at 30°C and treated with RNase. The radioactivity corresponding to [3H]RNA hybridized to the filters was measured after solubilization in Cellusolve. In order to correct for the efficiency of hybridization and loss of hybridizable sequences during the washing and RNase treatment step, we included an appropriate internal standard, [32P]cRNA transcribed from ovalbumin cDNA or from pOV2.4 or pOVl.8 DNA, in the hybridization mixture (15). The amount of [3H]RNA specifically hybridized to the filters was calculated as the cpm competed for in the presence of unlabeled RNA. Electrophoresis and Transfer of RNA to Diazobenzyloxylmethyl-Paper and Hybridization to DNA Probes. Oviduct nuclear RNA was isolated and subjected to agarose gel electrophoresis in the presence of methylmercury hydroxide as described (12). The RNA was transferred from the gel to diazobenzyloxylmethyl-paper and hybridized to [32P]DNA by a modification of the method of Alwine et al. (16) as described (12). pOV230 DNA was labeled with 32p to a specific activity of 1-3 X 108 cpm/,ug by nick translation (12). RESULTS [3H]RNA Synthesis In Vitro in Isolated Nuclei. Incorporation of radioactive nucleotides into trichloroacetic acidprecipitable material, which represents RNA synthesized de novo, reached a plateau in about 15 min in the isolated nuclei reaction mixture at 37°C (Table 1). The incorporation was rapid during the first 5 min, and addition of a 100-fold excess of nonlabeled UTP to the reaction mixture after 10 min showed no decrease in this value for the following 60 min, indicating that there was no major degradation of the newly synthesized [3H]RNA. It is likely that the majority of the RNA synthesis observed in the nuclei is due to the completion of chains initiated in vivo prior to the in vitro incubation and thus reflects Table 1. Effect of temperature and of various inhibitors on [3H]RNA synthesis in nuclei isolated from hormone-stimulated oviducts*
pmol [3H]UMP incorporated/
pg DNA at: Conditions
5 min
10 min
15 min
Reaction mixture at 370C + a-Amanitin (0.5, g/ml) + a-Amanitin (100;tg/ml) + 1 mM UTP after 10 min + Actinomycin D (40 pg/ml)
0.15 0.08 0.03 0.15 0.008
0.18 0.09 0.04 0.18 0.009
0.20 0.09 0.08 0.19 0.012
0.04 0.09 0.14 Reaction mixture at 250C + Actinomycin D (40,ug/ml) 0.002 0.004 0.008 * The assays contained 0.2 pg of nuclear DNA per ml. Values are averages of four determinations.
the transcriptional activity that remains in the nuclei after isolation. The time course of the [3H]RNA synthesis was linear for about 60 min when the incubation was performed at 250C. In nuclei from DES-stimulated oviducts (Table 1), 45-52% of the total [3H]RNA synthesis was sensitive to concentrations of a-amanitin (0.5 ,gg/ml) that inhibit transcription by polymerase II (17, 18). In the presence of concentrations of aamanitin (100,gg/ml) that inhibit polymerases II and III, about 20-38% of the activity remained, indicating that polymerase I also was active under the conditions used. In order to rule out the possibility of RNA-dependent [3H]RNA synthesis, actinomycin D (40 ,tg/ml) was added in the reaction mixture, and RNA synthesis was inhibited by 95%. Quantitation of Nascent Hormone-Induced [3H]mRNA0, Sequences. Table 2 shows the concentration of mRNAOv sequences present in the [3H]RNA synthesized by nuclei isolated from chicken oviduct under different states of estrogen stimulation. Nuclei from DES-stimulated chickens synthesized mRNA0v sequences at a concentration of z0.23% of the total [3H]RNA, while no detectable mRNAOv sequences were found in [3H]RNA synthesized in spleen and liver nuclei. This tissue-specific response represents a preferential synthesis in the sense that it corresponds to a substantial increase over the random transcription of the total chicken genome. The lack of mRNAO, synthesis in isolated nuclei from oviducts after withdrawal from hormonal treatment (Table 2) confirms the importance of estrogen in maintaining the synthesis of mRNAOv. Total [3H]RNA and [3H]mRNAov synthesis was inhibited about 96 and 97%, respectively, when actinomycin D (40 ,Ig/ml) was present in the reaction mixture. This inhibition appears to be random in that the total mass of newly synthesized [3H]RNA is greatly decreased (Table 1) but there is no preferential inhibition of [3H]mRNA., sequences since its concentration remains unaltered in the presence of actinomycin D (Table 2). These results are consistent with the idea that the synthesis of mRNAO, sequences is a DNA-dependent process. The fact that the hybridizable [3H]RNA can be competed for by mRNAOv indicates that these transcripts were indeed mRNAkv sequences (Table 2). Inhibition of the synthesis of mRNA0v by a-amanitin at low concentrations substantiates that they were transcribed
by RNA polymerase II.
Next we examined the effects of hormone withdrawal on the expression of the ovalbumin gene in oviduct nuclei. The decrease was rapid and appeared to be exponential (Fig. 1). Under these conditions, no accurate assessment of t1/2 of the response could be made because of the slow clearance of DES from the animals. After 60 hr there was no detectable mRNAOv in the transcripts, while the total [3H]RNA synthesis in nuclei was not altered greatly. This preferential loss of expression of the ovalbumin gene after 60 hr of hormone withdrawal was readily reversible within 1-2 hr after readministration of a single injection (secondary stimulation) of estrogen (Fig. 2). There was an acute increase in the synthesis of mRNAO, sequences, which reached a peak by 8 hr. When a second injection was given after 12 hr of secondary stimulation, the synthesis of mRNAO, increased to values approaching those of oviduct nuclei prepared from chickens chronically treated with DES (data not shown). Similarly, when chickens were withdrawn from hormone for periods of 72 or 86 hr, induction of mRNA0v synthesis in isolated nuclei was observed, but the maximal levels of induction were
suppressed (data not shown). Induction of Structural and Intervening Sequences in [3H]RNA0, Synthesized In Vitro. In DES-stimulated oviducts structural and intervening sequences of the ovalbumin gene are transcribed in vivo and in vitro (12). Here we have mea-
Biochemistry: Swaneck et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 2. Specificity of nuclear
1051
[:3H]mRNA0, synthesis in vitro from different tissues Hybridizable
Nuclei from
Oviduct DES*
+ (Y-Amanitin (0.5 pg/ml) + Actinomycin D (40 ug/ml)
Oviduct Wt 3 days
VHJRNA
[:32PlcRNA0,
gene
Input,
hybridized, cpm
recovery, %
sequences, cpm
cpm X 10-6
% of total RNA
1215 244
14.7
6950
2.92
0.238
mRNA(,
549 112
18.6
2350
1.02
0.231
mRNA(,
52 46
18.3
33
1.63
0.002
-
469
18.2
2576
1.1
0.234
-
121 131 99 103 62 87 33 29
17.1
0
1.05
Biochemistry
Effect of estrogen on gene expression in chicken oviduct: Evidence for transcriptional control of ovalbumin gene* (nuclear transcription/cloned DNA/hormone action/intervening gene sequences/precursor RNA)
GEORGE E. SWANECK, JEFFREY L. NORDSTROM, FRITZ KREUZALER, MING-JER TSAI, AND BERT W. O'MALLEY Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
Communicated by Sidney P. Colowick, November 27, 1978
The transcription of structural and intervening ABSTRACT se uences of the chicken ovalbumin gene was studied in nuclei isoated from the oviduct, liver, and spleen of chickens in different states of estrogen stimulation. The concentration of transcripts of structural and intervening DNA sequences was determined by hybridizing the newly synthesized [3HJRNA to filters containing cloned ovalbumin cDNA (pOV230) or fragments of the natural ovalbumin gene (pOV2.4 and pOV1.8). Of the RNA synthesized by oviduct nuclei from chickens chronically stimulated with diethylstilbestrol, 0.23% corresponded to ovalbumin mRNA and 0.17% were transcripts of intervening sequences. No detectable ovalbumin mRNA sequences were synthesized by nuclei from spleen and liver. After 60 hr of hormone withdrawal, synthesis of ovalbumin mRNA by oviduct nuclei could not be detected. After readministration of estrogen, a gradual increase in ovalbumin mRNA synthesis was observed which began at 1 hr and reached a plateau by 8 hr. For the intervening sequences, similar kinetics were observed for the initial 4 hr. Previously we had identified multiple species of putative precursors of ovalbumin mRNA in oviduct nuclei from chickens chronically stimulated with diethylstilbestrol. We demonstrate here that withdrawal of diethylstilbestrol resulted in a depletion of high-molecular-weight ovalbumin RNA and of mature ovalbumin mRNA and that readministration of the estrogen induced the nuclear accumulation of both forms of ovalbumin RNA. These findings indicate that: (i) a method exists to assay synthesis of hormone-inducible specific eukaryotic [3H]mRNA in vitro; (ii) the estrogen-mediated preferential expression of the ovalbumin gene is maintained in isolated oviduct nuclei; (iii) after hormone withdrawal, a single injection of diethylstilbestrol induces transcription of ovalbumin structural and intervening sequences, with nuclear accumulation of high-molecular-weight ovalbumin RNA and mature ovalbumin mRNA; and (iv) these results are consistent with regulation of ovalbumin mRNA at the level of ovalbumin gene transcription. Previous studies on the regulation of gene expression in eukaryotes have shown that steroid hormones induce synthesis of specific proteins in vivo by increasing the concentration of their mRNAs (1-3). Injections of estrogen into chickens that had been prestimulated with estrogen and then withdrawn from all hormone (secondary stimulation) induced the accumulation of ovalbumin mRNA (mRNAOv) (4-7). Recently, it has been shown that the structural sequences of the ovalbumin gene (8-10) and globin gene are interrupted by multiple regions of nonstructural intervening sequences. In addition, the 15S precursor to globin mRNA was shown to contain both structural and intervening sequences of the globin gene (11). Similarly, transcripts of both types of ovalbumin sequences accumulated in vivo in oviduct nuclei after stimulation with estrogen (12). In addition, we have demonstrated the existence of high-mo-
lecular-weight species of ovalbumin RNA in nuclear extracts of oviducts stimulated chronically with estrogen (12). Because these molecules contain sequences complementary to intervening as well as to structural sequences of the ovalbumin gene, it is likely that these RNAs are precursors to mature
mRNA0v.
In an attempt to define the mechanism of steroid hormoneinduced accumulation of mRNA0v, we have studied the rate of transcription of the ovalbumin gene in vitro and the size of the ovalbumin gene transcripts in vivo. In the present study, we report that secondary stimulation of chickens with diethylstilbestrol (DES) elevates the extent of transcription of both structural and intervening sequences of the ovalbumin gene as assayed in isolated oviduct nuclei. We also demonstrate that DES stimulation increases the level of high-molecular-weight ovalbumin sequence-containing RNA in vivo in addition to that of mature mRNA0v. These results further support the hypothesis that an increase in the rate of transcription of the ovalbumin gene appears to be the major mechanism by which estrogen elevates the intracellular level of mRNA., (4, 5). MATERIALS AND METHODS Hormone Treatment. Estrogen-stimulated oviducts were obtained from White Leghorn chickens that were implanted weekly with a 20-mg pellet of DES. This pellet provided continuous release of DES for 8-9 days. Estrogen-withdrawn oviducts were obtained from chickens that had received daily subcutaneous injections of 2.5 mg of DES for 14 days and were subsequently withdrawn from hormone for periods up to 14 days. For experiments involving acute stimulation with estrogen, one injection of 2.5 mg of DES was given to hormonewithdrawn chickens. Oviducts were collected at the time intervals indicated in the text. Synthesis and Isolation of [3HIRNA from Nuclei. Nuclei were purified from oviducts of either hormone-stimulated or withdrawn chickens as described (12). [3H]RNA was transcribed in isolated nuclei in a reaction mixture of 5 ml that contained 0.8 mM each of ATP, GTP, and UTP, 0.014 mM [3H]CTP (35.4 Ci/mmol, 1 Ci = 3.70 X 1010 Bq), 3 mM MgCI2, 2.5 mM MnCI2, 3 mM dithiothreitol, 0.5 mM EDTA, 100 mM KCI, 12.5% (vol/vol) glycerol, 50 mM Tris-HCl (pH 7.8), and 0.8-2.2 X 108 nuclei per ml. After 30 min at 37°C, the [3H]RNA synthesized was isolated by the procedure of Roop et al. (12). Abbreviations: DES, diethylstilbestrol; pOV230 DNA, plasmid DNA containing a nearly full-length ovalbumin cDNA insert (13); pOVl.8 DNA, plasmid DNA containing the 1.8-kilobase EcoRI fragment of the natural ovalbumin gene (14); pOV2.4 DNA, plasmid DNA containing the 2.4-kilobase EcoRI fragment of the natural ovalbumin gene (14); mRNAov, ovalbumin mRNA. * This is paper no. 22 in a series of publications from our laboratory dealing with hormone regulation of gene expression in the chicken oviduct. Paper no. 21 is ref. 15.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1049
1050
Proc. Natl. Acad. Sci. USA 76 (1979)
Biochemistry: Swaneck et al.
Hybridization of [3HJRNA to DNA Filters. Filters containing pOV230, pOV1.8, or pOV2.4 DNA were prepared as described (9, 12-14). All preparations of cloned DNA were performed in a P3 facility and all procedures were carried out in accordance with National Institutes of Health guidelines. Ovalbumin structural and intervening sequence transcripts were measured by hybridizing the in vitro [3H]RNA to nitrocellulose filters containing pOV230 or pOV2.4 or pOVl.8 DNA as described (12). Briefly, 1-4 X 106 cpm of [3H]RNA was hybridized to DNA filters (1.2 ,ug) at 420C for 18 hr in 50% (vol/ vol) formamide/0.21 M NaCl/0.021 M Na citrate at pH 7.0 in the presence and absence of competitor (7.5 ,tg of unlabeled mRNAOv or 9 ,ug of unlabeled cRNA transcribed from pOV2.4 or pOVl.8 DNA). At the end of incubation, the filters were extensively washed in 0.30 M NaCl/0.030 M Na citrate at pH 7 at 30°C and treated with RNase. The radioactivity corresponding to [3H]RNA hybridized to the filters was measured after solubilization in Cellusolve. In order to correct for the efficiency of hybridization and loss of hybridizable sequences during the washing and RNase treatment step, we included an appropriate internal standard, [32P]cRNA transcribed from ovalbumin cDNA or from pOV2.4 or pOVl.8 DNA, in the hybridization mixture (15). The amount of [3H]RNA specifically hybridized to the filters was calculated as the cpm competed for in the presence of unlabeled RNA. Electrophoresis and Transfer of RNA to Diazobenzyloxylmethyl-Paper and Hybridization to DNA Probes. Oviduct nuclear RNA was isolated and subjected to agarose gel electrophoresis in the presence of methylmercury hydroxide as described (12). The RNA was transferred from the gel to diazobenzyloxylmethyl-paper and hybridized to [32P]DNA by a modification of the method of Alwine et al. (16) as described (12). pOV230 DNA was labeled with 32p to a specific activity of 1-3 X 108 cpm/,ug by nick translation (12). RESULTS [3H]RNA Synthesis In Vitro in Isolated Nuclei. Incorporation of radioactive nucleotides into trichloroacetic acidprecipitable material, which represents RNA synthesized de novo, reached a plateau in about 15 min in the isolated nuclei reaction mixture at 37°C (Table 1). The incorporation was rapid during the first 5 min, and addition of a 100-fold excess of nonlabeled UTP to the reaction mixture after 10 min showed no decrease in this value for the following 60 min, indicating that there was no major degradation of the newly synthesized [3H]RNA. It is likely that the majority of the RNA synthesis observed in the nuclei is due to the completion of chains initiated in vivo prior to the in vitro incubation and thus reflects Table 1. Effect of temperature and of various inhibitors on [3H]RNA synthesis in nuclei isolated from hormone-stimulated oviducts*
pmol [3H]UMP incorporated/
pg DNA at: Conditions
5 min
10 min
15 min
Reaction mixture at 370C + a-Amanitin (0.5, g/ml) + a-Amanitin (100;tg/ml) + 1 mM UTP after 10 min + Actinomycin D (40 pg/ml)
0.15 0.08 0.03 0.15 0.008
0.18 0.09 0.04 0.18 0.009
0.20 0.09 0.08 0.19 0.012
0.04 0.09 0.14 Reaction mixture at 250C + Actinomycin D (40,ug/ml) 0.002 0.004 0.008 * The assays contained 0.2 pg of nuclear DNA per ml. Values are averages of four determinations.
the transcriptional activity that remains in the nuclei after isolation. The time course of the [3H]RNA synthesis was linear for about 60 min when the incubation was performed at 250C. In nuclei from DES-stimulated oviducts (Table 1), 45-52% of the total [3H]RNA synthesis was sensitive to concentrations of a-amanitin (0.5 ,gg/ml) that inhibit transcription by polymerase II (17, 18). In the presence of concentrations of aamanitin (100,gg/ml) that inhibit polymerases II and III, about 20-38% of the activity remained, indicating that polymerase I also was active under the conditions used. In order to rule out the possibility of RNA-dependent [3H]RNA synthesis, actinomycin D (40 ,tg/ml) was added in the reaction mixture, and RNA synthesis was inhibited by 95%. Quantitation of Nascent Hormone-Induced [3H]mRNA0, Sequences. Table 2 shows the concentration of mRNAOv sequences present in the [3H]RNA synthesized by nuclei isolated from chicken oviduct under different states of estrogen stimulation. Nuclei from DES-stimulated chickens synthesized mRNA0v sequences at a concentration of z0.23% of the total [3H]RNA, while no detectable mRNAOv sequences were found in [3H]RNA synthesized in spleen and liver nuclei. This tissue-specific response represents a preferential synthesis in the sense that it corresponds to a substantial increase over the random transcription of the total chicken genome. The lack of mRNAO, synthesis in isolated nuclei from oviducts after withdrawal from hormonal treatment (Table 2) confirms the importance of estrogen in maintaining the synthesis of mRNAOv. Total [3H]RNA and [3H]mRNAov synthesis was inhibited about 96 and 97%, respectively, when actinomycin D (40 ,Ig/ml) was present in the reaction mixture. This inhibition appears to be random in that the total mass of newly synthesized [3H]RNA is greatly decreased (Table 1) but there is no preferential inhibition of [3H]mRNA., sequences since its concentration remains unaltered in the presence of actinomycin D (Table 2). These results are consistent with the idea that the synthesis of mRNAO, sequences is a DNA-dependent process. The fact that the hybridizable [3H]RNA can be competed for by mRNAOv indicates that these transcripts were indeed mRNAkv sequences (Table 2). Inhibition of the synthesis of mRNA0v by a-amanitin at low concentrations substantiates that they were transcribed
by RNA polymerase II.
Next we examined the effects of hormone withdrawal on the expression of the ovalbumin gene in oviduct nuclei. The decrease was rapid and appeared to be exponential (Fig. 1). Under these conditions, no accurate assessment of t1/2 of the response could be made because of the slow clearance of DES from the animals. After 60 hr there was no detectable mRNAOv in the transcripts, while the total [3H]RNA synthesis in nuclei was not altered greatly. This preferential loss of expression of the ovalbumin gene after 60 hr of hormone withdrawal was readily reversible within 1-2 hr after readministration of a single injection (secondary stimulation) of estrogen (Fig. 2). There was an acute increase in the synthesis of mRNAO, sequences, which reached a peak by 8 hr. When a second injection was given after 12 hr of secondary stimulation, the synthesis of mRNAO, increased to values approaching those of oviduct nuclei prepared from chickens chronically treated with DES (data not shown). Similarly, when chickens were withdrawn from hormone for periods of 72 or 86 hr, induction of mRNA0v synthesis in isolated nuclei was observed, but the maximal levels of induction were
suppressed (data not shown). Induction of Structural and Intervening Sequences in [3H]RNA0, Synthesized In Vitro. In DES-stimulated oviducts structural and intervening sequences of the ovalbumin gene are transcribed in vivo and in vitro (12). Here we have mea-
Biochemistry: Swaneck et al.
Proc. Natl. Acad. Sci. USA 76 (1979)
Table 2. Specificity of nuclear
1051
[:3H]mRNA0, synthesis in vitro from different tissues Hybridizable
Nuclei from
Oviduct DES*
+ (Y-Amanitin (0.5 pg/ml) + Actinomycin D (40 ug/ml)
Oviduct Wt 3 days
VHJRNA
[:32PlcRNA0,
gene
Input,
hybridized, cpm
recovery, %
sequences, cpm
cpm X 10-6
% of total RNA
1215 244
14.7
6950
2.92
0.238
mRNA(,
549 112
18.6
2350
1.02
0.231
mRNA(,
52 46
18.3
33
1.63
0.002
-
469
18.2
2576
1.1
0.234
-
121 131 99 103 62 87 33 29
17.1
0
1.05
