Effect of estradiol on rat uterus DNA-dependent RNA polymerases. Studies on solubilized enzymes.

DNA-dependent RNA polymerases of immature and castrated rat uteri were studied after estradiol administration. The enzymes were solubilized from either whole uterus homogenate or nuclei and their activities were measured on an exogenous DNA template. alpha-Amanitin was used to distinguish alpha-amanitin-resistant from alpha-amanitin-senitive forms of the enzyme. The number of alpha-amanitin-sensitive RNA polymerase molecules was measured by a binding assay using labeled amanitin. In the first series of experiments RNA polymerases were solubilized from whole uterus homogenate. alpha-Amanitin-sensitive and -resistant activities were constant during the first 6 hours after estradiol treatment, followed by a late and moderate increase in their activities (50% at 12 hours for the resistant form and 40% at 24 hours for the sensitive form). The number of sensitive polymerase molecules evolved in an identical manner to its activity (+40% at 24 hours), suggesting that the increase in activity is due to the synthesis of new enzyme molecules. For both forms, no diffusible stimulatory factor was detected in the uterus of hormone-treated animals. In the second series of experiments, disrupted nuclei were washed with 0.15 M (NH4)2SO4 in order to release only enzyme molecules which were not firmly bound to DNA in a transcription complex. The amount of the sensitive form of polymerase which remains firmly bound to chromatin, was constant for 6 hours after estradiol administration and was doubled by 24 hours. The firmly bound alpha-amanitin-resistant activity was solubilized and was measured in the presence of an exogenous template. There was a progressive increase in activity first detectable in 1 to 2 hours, amounting to 50% at 6 hours and 100% at 24 hours. The reported results show that during the first 6 hours of hormone treatment: (a) the total content of RNA polymerases remains unchanged in the uterus; (b) the number of alpha-amanitin resistant molecules tightly bound to DNA increases progressively while the alpha-amanitin sensitive remains constant. At a later time (24 hours), an increase is observed both for the total amount of enzymes and for their fraction engaged in a transcription complex.

DNA-dependent RNA polymerases of immature and castrated rat uteri were studied after estradiol administration.
The enzymes were solubilized from either whole uterus homogenate or nuclei and their activities were measured on an exogenous DNA template. oc-Amanitin was used to distinguish Lu-amanitin-resistant from cu-amanitin-senitive forms of the enzyme. The number of oc-amanitin-sensitive RNA polymerase molecules was measured by a binding assay using labeled amanitin.
In the first series of experiments RNA polymerases were solubilized from whole uterus homogenate. cY-Amanitin-sensitive and -resistant activities were constant during the first 6 hours after estradiol treatment, followed by a late and moderate increase in their activities (50% at 12 hours for the resistant form and 40% at 24 hours for the sensitive form). The number of sensitive polymerase molecules evolved in an identical manner to its activity (+40% at 24 hours), suggesting that the increase in activity is due to the synthesis of new enzyme molecules. For both forms, no diffusible stimulatory factor was detected in the uterus of hormone-treated animals. In the second series of experiments, disrupted nuclei were washed with 0.15 M (NH,),SO, in order to release only enzyme molecules which were not firmly bound to DNA in a transcription complex. The amount of the sensitive form of polymerase which remains firmly bound to chromatin, was constant for 6 hours after estradiol administration and was doubled by 24 hours. The firmly bound uc-amanitinresistant activity was solubilized and was measured in the presence of an exogenous template. There was a progressive increase in activity first detectable in 1 to 2 hours, amounting to 50% at 6 hours and 100% at 24 hours.
The reported results show that during the first 6 hours of hormone treatment: (a) the total content of RNA polymerases remains unchanged in the uterus; (b) the number of a-amanitin resistant molecules tightly bound to DNA increases progressively while the cu-amanitin sensitive remains constant. At a later time (24 hours), an increase is observed both for the total amount of enzymes and for their fraction engdged in a transcription complex.
Increase of RNA polymerase activities measured in vitro have been reported in isolated nuclei of uterus of ovariectomized or immature rats in the first hours following estradiol treatment (l-6). Due to the complexity of the system in which RNA synthesis is dependent on the endogenous template, interpretation of the data is difficult. In fact, the observed modifications can be the result of several phenomena which may eventually act simultaneously. (a) A change in the avail-* Present address, Roussel Uclaf, B.P. no 9, 93230-Romainville, France.
ability of the chromatin template; this was already investigated using bacterial RNA polymerase and a 25 to 50'7 increased RNA synthesis was observed as early as 1 hour after hormone treatment (6-10). (b) A change in the number of active RNA polymerase molecules; this could be related to a change in the actual number of enzyme molecules or to a change in the activities of preexisting enzyme molecules (by covalent modification or interaction with a control "factor").
In an attempt to distinguish between these possibilities, the variations in RNA polymerase activities solubilized from  (16).

RNA Polymerase
Solubilization-All steps were carried out at 0" as rapidly as possible. Frozen uteri (0.5 to 1 g) were pulverized in liquid nitrogen with the help of a mortar and homogenized with a glass-class homogenizer in 14 mlof MS (25) buffer and aliquots were taken for the measurement of DNA and 13H lamanitin binding to RNA nolvmerases B. The remainder of the homogenate was dilutedto 20 ml with MS(20) buffer containing 0.8 M ammonium sulfate. The viscous mixture, maintained below 0" in a NaCl-ice bath, was sonicated four or five times at the lowest energy for 10-s periods. The temperature was controlled between each period and was at all times below 5". The sonicate was centrifuged at 20,000 x g for 10 min in a Sorvall centrifuge and the supernatant was precipitated by ammonium sulfate (50% saturation).
After 30 to 45 min the precipitate was collected by centrifugation at 200,000 x g for 30 min in a Spinco SW 41 rotor. The pellet was suspended in 5 ml of MS(50) buffer (P50 fraction) and the ammonium sulfate concentration was determined by conductivity measurement after a l,OO+fold dilution. This extract can be stored in liquid nitrogen for several months without any loss of activity. Preparation of Nuclear Fraction-Uteri were homogenized in 6 volumes of 0.32 M sucrose/l mM MgCl, by 10 strokes in a glass-glass homogenizer.
The homogenate was centrifuged at 800 x g for 10 min and the nuclear pellet washed twice with the same medium.
The nuclei were disrupted in MS (25) (12). Effects of Estradiol-The amount of enzyme B in the uterus was measured on the uterus homogenate at different times after injection of estradiol to immature and castrated rats. In order to express the results on a cellular basis, the amount of enzyme B was related to the DNA content of the organ. The base-line values for the immature rat (4 pmol/mg of DNA) is 50% higher than that of the castrated animal (2.6 pmol/mg of DNA) (Fig. 3). The calculated number of polymerase B molecules per haploid genome is of the order of 8 x lo3 for the immature and 5 x lo3 for the castrated animal. This value is 4 to 5 times lower than that found for the uterus and other tissues of the normal adult rat (12). The variation in the amount of enzyme B as a function of time after a single estradiol injection is shown in Fig. 3. The amount of enzyme per cell remains constant over the first 12 hours and then increases by 40$ in both the immature and castrated animals at 24 hours. Since at this time the protein content of the uterus is also increased, the amount of enzyme B molecules per mg of protein is increased by only 10 to 15%.  Table I indicate an almost total recovery after sonication followed by centrifugation. After precipitation, at and above I 50% ammonium sulfate, the recovery of enzyme B was more than 80%. For each series of experiments, the recovery of enzyme B was checked by measuring the amount present in the intial homogenate and in the P50 fraction. The recovery varied between different series of animals (65 to 80%) but within a given series, it is fairly constant. As seen in Table II, the solubilized enzyme activity exhibits all of the characteristics of DNA-dependent RNA polymerases. The effect of cY-amanitin concentration on the activity of solubilized RNA polymerase is shown in Fig. 4. This inhibitor distinguishes enzyme A activity cu-amanitin-resistant, from enzyme B activity, n-amanitinsensitive.
At the concentration of inhibitor (1 to 10 PM) used in these experiments, enzyme C activity, is measured along with enzyme A activity.  FIG. 4. Effect of a-amanitin on the activity of solubilized RNA polymerase B-The activity of solubilized enzymes (P50 fraction) prepared from estradiol-treated (24 hours) immature rat uteri was measured under standard conditions (see "Materials and Methods") except that a-amanitin was present in the incubation medium at the indicated concentration.
The effects of divalent cation concentration and ionic strength on the solubilized enzymatic activities are presented in Fig. 5. In the presence of 4 mM Mn*+, the optimum concentration of ammonium sulfate was 40 mM for enzyme A and 100 mM for enzyme B. With 40 mM ammonium sulfate, Mg*+ and Mn*+ at their optimum concentration were equally effective for enzyme A. At this concentration of ammonium sulfate, Mn*+ was a better activator (2-fold) than Mg*+ for enzyme B. These differences between enzymes A and B are in agreement with those reported for purified enzymes of calf thymus (19) and rat liver (20).
The K, for UTP of the total enzyme (A + B) was 0.03 mM, a value similar to that obtained for purified calf thymus enzymes (11). Based on this value and those reported for the other three nucleotides with pure enzymes (ll), concentrations of 0.3 mM UTP and 2 mM for the other nucleotides were used in our assay. The kinetics of the reaction are shown in Fig. 6. The incorporation of [3H]UMP is linear for the first 6 min. Very often after this time, there was a decrease in the rate of RNA synthesis and the nucleotide incorporation was no longer linear. Consequently, only short incubation times (6 min) were used for these assays, in order to measure a maximum initial velocity. When the protein concentration in the incubation mixture was lower than 2 mg/ml, a linear relationship was observed between initial velocity and the amount of enzyme incubated (not shown). The presence of RNase activities was checked in the enzyme preparations in the following way. At the end of [3H]UMP incorporation, 20 rg of actinomycin D was added to the reaction mixture and the incubation continued for an additional 10 min at 37". As seen in Fig. 6 no RNase activity was detected in the P50 fraction. Occasionally a small decrease in the amount of synthetized RNA was observed, but it was always less than 10%.
Effects of Estrudiol-The variation of solubilized :NA polymerase A and B activities were studied in three seri s of immature and three series of castrated animals for time periods up to 24 hours, after a single injection of estradiol. As seen in Fig. 7, the basal activities of the immature animals were about 60% higher than those of the castrated group. In the control animals of both groups, enzyme B activity was greater than that of enzyme A. Under the influence of estradiol, enzyme A activity remains unchanged up to 6 hours. The first increase (50%) was observed after 12 hours and reached 100% by 24 hours. In the case of enzyme B, the increase in activity occurred later (24 hours) and was less pronounced (40%) than that observed for enzyme A at this time.
It was not possible to measure the recovery of enzyme A during the solubilization procedure. However, it should be pointed out that at a given time after hormone injection, the ratio of A to B activity was always the same in different experiments.
After 24 hours, the increase in enzyme B activity is the same (40%) as the increase in the amount of enzyme B measured by ['Hlamanitin binding (Fig. 3), indicating that the enhanced B activity corresponds to a higher number of enzyme molecules. In the case of enzyme A, it is not possible to know whether the change in activity observed after 12 hours is due to an increase in the number of active molecules or to a higher enzyme specific activity. That this increased activity is not due to the stimulation of preexisting molecules by a diffusible factor was suggested by the following experiment.
The enzymes were solubilized from uteri of control and 24.hourtreated immature animals. Enzyme A activity was measured in each preparation and in mixtures of both extracts. Table  III shows that the activity of a mixture of the two enzyme preparations is equal to the sum of the activities of each fraction. This strict additivity excludes the presence of an available diffusible stimulatory factor(s) in the extract prepared from uteri of estradiol-treated animals. During precipitation by ammonium sulfate, two fractions are discarded: the dialyzable components and the proteins which do not precipitate at 50% ammonium sulfate. These two fractions were prepared from three batches of castrated uteri (control, 6 hour-and 24-hour-treated), in the following way. Part of the uteri was processed as usual and the ammonium sulfate supernatant was dialyzed and concentrated against MS(O) buffer to obtain Fraction I. Another part was homogenized and dialyzed against distilled water. The concentrated dialysate is designated as Fraction II. RNA polymerase activities of the three enzyme preparations were then measured as usual and in the presence of an aliquot of the homologous fractions I, II, and I + II at concentrations corresponding to their respective tissular proportions.
For the three batches of uteri, a similar nonspecific 30% decrease in activity was observed, regardless of the fraction added (result not shown). These experiments suggest that no component, which could affect RNA polym-   and 30%, respectively, of the activity found in the total tissue (Fig. 8). Fig. 9 shows the variation of tightly bound enzyme A and B, after administration of estradiol. The number of enzyme B molecules bound to the chromatin remains constant during the first 6 hours and shows a 100% increase at 24 hours. The results are different for enzyme A. There is a progressive increase in bound enzyme A activity, already measurable at 1 hour (20%') and which reaches 80% at 24 hours. DISCUSSION RNA polymerases A (I) and B (II) were studied in estradioltreated rat uteri, either in whole tissue homogenate or in nuclei.
In whole tissue homogenate, similar results were obtained for castrated and immature rats. The amount of enzyme B molecules measured by 13H]amanitin binding in crude extracts, as well as the enzyme B activity measured after solubilization, remained constant over the first 12 hours after hormonal treatment and then showed a 40% increase at 24 hours. This strict correlation between the number of enzyme B molecules determined by amanitin binding and the enzymatic activity clearly shows that the delayed enhancement of activity is related to the synthesis of new B enzyme molecules. As previously discussed (12), this strict correlation makes also very unlikely the possibility that amanitin binding assays would measure the binding of the labeled toxin to free enzyme subunit(s) or enzymatic inactive B molecules which would be present in the cellular homogenates. Enzyme A activity solubilized from whole tissue did not change within the first 6 hours and then rose progressively (50% at 12 hours, 100% at 24 hours). However, the experiments did not differentiate between an actual increase in the number of enzyme A molecules and an activation of pre-existing enzyme molecules via nondiffusible macromolecular factor(s) present in limiting amount or a covalent modification of the enzyme. In this respect, it is worth mentioning that, for both enzymes, no dialyzable or macro-molecular stimulatory "factor(s)" was detected.
In a second series of experiments, isolated nuclei were washed with 0.15 M ammonium sulfate in order to eliminate any readily extractible RNA polymerases, while keeping enzyme molecules tightly bound to the chromatin. Since increasing the ionic strength prevents initiation of RNA synthesis and results in the release from the DNA template of RNA polymerase molecules which are not engaged in a ternary transcription complex' (23)(24)(25)(26), it is very likely that the enzyme activity which remains tightly bound to the chromatin corresponds to the fraction which is actively engaged in transcription in the form of enzyme.DNA.RNA ternary complexes. The number of enzyme B molecules tightly bound to the chromatin measured by [$H Jamanitin binding was constant over the first 6 hours following estradiol administration. This suggests a constant number of enzyme B molecules engaged in the process of transcription, but does not preclude any changes in the elongation rate of RNA chains or any qualitative changes in the nature of the template being transcribed. In contrast, there is a progressive increase in the amount of enzyme A tightly bound to the chromatin. This increase is already evident (20%) at 1 hour, reaching 45% at 6 hours. During this period, the enzyme A activity which can be extracted from total tissue and which represents the sum of readily extractible and tightly bound enzyme activities, remained constant. It therefore seems that, under the influence of hormone treatment, a portion of the enzyme A, which was originally present in a free form, becomes engaged in RNA synthesis. It remains to be seen whether this phenomenon is due to an increased availability of the nucleolar chromatin or to the effect of a factor which would be required for initiation of RNA synthesis.
It is interesting to compare the results obtained with solubilized RNA polymerases to those obtained when measuring the same enzyme activities in whole nuclei, under conditions where the enzymes are transcribing their own template. Such studies are reported elsewhere.' They led to the conclusion, which differs to some extent from that reached by other author(s) (6), that there was an early increase in the number of transcribing A enzyme molecules, while the number of transcribing B molecules was unchanged. Our present results fully support this interpretation.
In uiuo RNA metabolism has been extensively studied in the mouse and rat uterus. After administration of estradiol, an increase in total uterine RNA has been observed after 6 hours of hormone treatment (27)(28)(29)(30). The results are however conflicting when early quantitative and qualitative changes in RNA are studied by the incorporation of radioactive RNA precursors. After hormone treatment, some reports showed an early (2 min to 1 hour) increase of RNA specific activity (28,29,31,32) and in others, this increase was not seen before 6 hours (30,33). In addition it is not clear if this is related to a general increase of incorporation of precursors into all RNA species (33)(34)(35)(36)(37) or to an increase limited to either pre-ribosomal (38) or heterogenous high molecular weight RNA (38,39). Although no clear conclusion can be drawn at the present time from these in uiuo studies, it is interesting to note that the progressive increase in the tightly bound polymerase A activity which we have observed in the first hours after hormone injection agrees with that reported for ribosomal RNA synthesis (33,40). In addition, the delayed enhancement in the amount of enzyme B molecules tightly bound to chromatin may be corre-