THE JOURNAL OF BIOI.OG~CAL CHEMISTRY Vol. 252, No. 24, Issue of December 25, pp. 8812-9820, 1977

Printed in U.S.A.

Effects of the Steroid Sex Hormone, Antheridiol, on the Initiation of RNA Synthesis in the Simple Eukaryote, Achlya ambisexualis*

(Received for publication, June 8, 1977)

ROBERT B. SUTHERLAND+ AND PAUL A. HORGEN§

From the Department of Botany, University of Toronto, Erindale Campus, Mississauga, Ontario L5L 1 C6, Canada

The interaction of Escherichia coli RNA polymerase (ri- bonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6) with Achlya ambisexualis DNA and chromatin has been examined to measure the number of RNA polymerase binding and initiation sites. The rifampicin challenge assay was utilized to make these measurements. The number of high affinity polymerase binding sites at saturation was calculated from the number of enzyme molecules bound to DNA or to Achlya chromatin. The number of rifampicin- resistant initiation sites was determined from the total levels and the number of average chain length of RNA synthesized under conditions where only RNA polymerase in a previous stable preinitiation complex could function in the presence of the drug. Measurements were made to determine that RNA chain initiation was not due to initia- tion at nicked or end regions of the DNA.

Antheridiol administration to growing cultures of Achlya results in an increase in the chromatin template activity. Chromatin prepared from either vegetatively growing or hormone-treated cultures of Achlya both supported a con- stant RNA chain elongation rate and a chain size of approx- imately 575 nucleotides. These rates did not change follow- ing antheridiol treatment. Conversely, within 1 h after the addition of antheridiol, the concentration of RNA polymer- ase needed to saturate chromatin binding sites was in- creased by 62% in comparison to control values; by 4 h the level of polymerase bound to chromatin was twice that of non-hormone-treated control Achlya chromatin.

Associated with the increase in chromatin-bound RNA polymerase was an increase in the rifampicin-resistant initiation sites. Chromatin from non-hormone-treated Ach- lya initiated 8,000 RNA chainslpg of DNA, while chromatin from Achlya that had been exposed 4 h to antheridiol initiated 23,000 chainslpg of DNA. Nearly 50% of the DNA from Achlya unstimulated chromatin is available to bind RNA polymerase. This value increases to 64% when the chromatin is isolated from cultures that had been exposed 4

* This study was supported by National Research Council of Canada Grant A6938. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “Wuertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada.

8 To whom reprint requests should be made.

h to antheridiol. These data demonstrate that the antheri- diol-induced increase in transcriptive activity was due to an increased number of RNA polymerase binding and initiation sites on the chromatin.

The fungal genus Achlya (class Oiimycetes) represents the most primitive group of eukaryotic organisms which is known to respond to steroid sex hormones (1). In the presence of antheridiol, a sterol secreted by female strains of Achlya, male strains are stimulated to produce sex organ initials (antheridial branches) which eventually differentiate into male sex organs (1, 2). The hormone is active when assayed with Achlya ambisexualis (strain E87) in extremely low concentrations (6 x lo-‘* g/ml) (1). The addition of antheridiol to undifferentiated cultures also elicits several changes in macromolecular syntheses (3-6). Silver and Horgen (4) re- ported that antheridiol stimulated the synthesis of total cellu- lar RNA, poly(A+) mRNA, and protein. Furthermore, under defined hormone conditions, there is a temporal separation between the synthesis of ribosomal RNA and the synthesis of poly(A+) RNA and protein (mRNA synthesis and protein synthesis occurring immediately prior to sex organ initial formation) (4, 7). In a study utilizing somewhat different hormonal conditions. Timberlake (5) reported that antheridiol increased the rates of synthesis of rRNA, poly(A+) RNA, and protein. In addition, both Silver and Horgen (4) and Timber- lake (5) suggest that qualitative changes in RNA synthesis induced by antheridiol are responsible for the production of new proteins required for differentiation of the male sex organs. Associated with the hormonal effects on RNA and protein synthesis, there also exists a corresponding change in the composition of the nuclear chromosomal proteins. Imme- diately prior to the observed enhancement of poly(A+) RNA synthesis, there is acetylation of specific “histone-like” pro- teins in Achlya (8).

It has been suggested that a key gene product that is accumulated during hormonal-induced differentiation in Ach- lya is the enzyme cellulase (EC 3.2.1.4) (3, 9, 10). Recently, it has been shown that antheridiol stimulated the accumulation of an “induced protein” with a molecular weight of 69,000 (6). Further characterization of the induced protein will certainly indicate whether it is indeed cellulase or perhaps some other key enzymatic or structural protein.

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Antheridiol Regulation of Transcription in Achlya 8813

Because of its phylogenetic simplicity, the study of hor- monal effects on differentiation in Achlya will no doubt shed some light upon how specific “effector molecules” regulate complex developmental processes in simple eukaryotic sys- tems. Since Achlya is an extremely fast growing eukaryotic microbe (111, which is easy to manipulate, susceptible to genetic alterations (12), and can be induced to differentiate in a highly uniform manner (entire thallus responds as target tissue to hormonal stimulation), many workers are interested in utlizing Achlya as a “model system” useful in ellucidating the general effects of steroid hormones on all aspects of cellular metabolism (2-13).

In more complex eukaryotes, the induction of specific struc- tural proteins by steroid hormones is mediated by changes in transcription (14-18). In animal systems, estrogen stimulates the de nouo synthesis of messenger RNA coding for ovalbu- min, while progesterone affects the synthesis of avidin mes- senger RNA (14). Prior to the increase in steroid hormone- induced protein synthesis, dramatic changes were reported for the levels of nuclear RNA (X1-17), RNA polymerase activity (18, 191, chromatin template activity (18, 201, and chromatin function (21). Evidence exists which suggests that hormones may function by elevating chromatin template capacity (22, 23).

Methods have been developed which allow workers to mea- sure the number of RNA synthetic initiation sites on eukar- yotic chromatin isolated from different developmental stages of hormone-stimulated target tissue (24, 25). These methods utilize eukaryotic chromatin, Escherichia coli RNA polymer- ase, and the antibiotic rifampicin. Tsai et al. (26) have shown that when transcribing chromatin in vitro, homologous hen oviduct RNA polymerase II and E. coli RNA polymerase holoenzyme compete for the same initiation sites. Their re- sults demonstrated that the efficiency of transcribing a specific gene depends on the interaction between the RNA polymerase (homologous or heterologous) and the chromatin elements of the initiation region (25, 26). Consequently, it has been suggested that steroid hormones affect transcription by mak- ing more areas of the genome free to bind RNA polymerase and be transcribed (24-26).

Do similar mechanisms work in more primitive eukaryotic systems? We have investigated the effects of antheridiol on the transcriptional ability of chromatin isolated from Achlya at selected times during the early stages of male sex organ development. We have also examined the initiation of RNA synthesis on Achlya DNA and chromatin utilizing E. coli RNA polymerase and the antibiotic rifampicin. The results reported in this study show that antheridiol dramatically increases the quantity of binding and initiation sites for RNA polymerase on Achlya chromatin.

MATERIALS AND METHODS

Materials -Escherichia coli RNA polymerase (Fraction IV con- taining Sigma factor), unlabeled nucleoside triphosphates, Tween 80, RNase-free sucrose, and ribonuclease A were purchased from Sigma. Sodium lauryl sulfate and proteinase K were obtained from BDH Chemicals. 13HlUTP and PCS scintillation fluid obtained from Amersham/Searle. Rifampicin was obtained from Calbiochem. Me- dia components were obtained from Difco. All other chemicals were reagent grade or better and were purchased from BDH Chemicals, J. T. Baker Chemical Co., or Fisher Scientific Co. Glass-distilled H,O was used for making all solutions.

Culture Maintenance and Growth-Achlya ambisexualis (strain E87dl was maintained on Emerson’s YPSS (Difco) agar slants. The mycelium was subcultured on PYG (1.25 g of yeast extract, 1.25 g of peptone, 3.0 g of glucose/liter of distilled H,O) agar plates for spore

preparation according to the method of Griffin and Breuker (271. Spore cysts were then inoculated into PYG broth, grown for 24 h at 24 + 1” on a rotarv shaker (130 rnml until a tinelv susnended mycelia had formed.“This was used immediately or stored at 4 f 1” for no longer than 48 h. Of this finely suspended mycelia, 300 ml were inoculated into 10 liters of mating medium (28). The cultures were grown for 48 h at 23 ? 1”. At this time, the cultures either received antheridiol at 5 x lo-ii g/ml (106 PM) or were left to grow without hormone.

Preparation of Nuclei and Chromatin - Mycelia from carboys were harvested by filtration through Miracloth and washed exhaustively with glass-distilled water. The excess H,O was gently squeezed from the mycelia and weight measurements were taken. Then 100 to 200 g, fresh weight, of Achlya were suspended in 2 volumes of nuclear isolation buffer (0.5 M sucrose, 10% elvcerol, v/v, 2% Tween- 80, v/v, 2.5 mM MgCl,, 0.5 mM dithiothreitoi, h.5 m& phenylmethyl- sulfonvl fluoride, and 10.0 mM Tris/HCl, pH 7.9). The suspended mycelia were then homogenized for 30 s at-the highest setting of a Willems Polytron Homogenizer (Brinkman Instruments).

The homogenate was filtered throueh 2 lavers of Miracloth and - - centrifuged at 4080 x g for 20 min. The crude nuclear pellet was then suspended in 0.14 M NaCl, 5.0 rnsr MgCl,, 10.0 mM Tris/HCl, pH 7.6, and centrifuged at 5000 x g for 10 min. The pellet was resuspended in the NaCl buffer and centrifuged again at 5000 x g for 16min. Chromatin was isolated followinathe orocedure of Rizzo

I I

and Nooden (29). The nuclear pellet was suspended in lysis buffer (10.0 mM Tris-HCl. DH 8.0. 1.0 rnM EDTA) and sonicated with a Bronwill Biosonik ilk (5 x IO s bursts at setting 60). This sonicated lysate was centrifuged at 4080 x g for 15 min. The pellet was resuspended in a small quantity of the lysis buffer and centrifuged again. The supernatants from the last two centrifugations were combined and made to 10 mM CaCl, with solid salt. This was mixed thoroughly and allowed to stand on ice for 15 min. The mixture was then centrifuged at 6000 x g for 15 min to pellet the chromatin.

Since histones are known to be extremely sensitive to proteolytic enzymes (30) and since it has been demonstrated that Achlya has “histone-like” chromosomal proteins (31), the possibility of proteo- lytic degradation of chromatin must be considered when interpreting experiments dealing with template availability. Basic chromosomal proteins were monitored for degradation by polyacrylamide gel electrophoresis according to the procedure of Panyim and Chalkley (32). The basic chromosomal proteins isolated from nuclei were similar to the histone-like proteins recovered from chromatin and the gel profiles suggested no evidence for discernible differences in protein degradation between the two fractions.

Analysis of Chromatin Components-Protein content was esti- mated by the method of Lowry et al. (33). DNA determinations were made by first incubating an aliquot of chromatin with 1 mg/ml of RNase A at 37” for 60 min. To this mixture 500 pg/ml of proteinase K were added and incubated for another 60 min at 26”. DNA was orecinitated bv making the solution 5% (v/v) with trichloroacetic acid and the mixture was then centrifuged at 8000 x g for 10 min. The DNA precipitate was suspended in 0.3 M perchloric acid and heated at iOOO for 10 min. DNA determinations were made by the diphenylamine method (34) using purified Achlya DNA as a stan- dard. RNA was estimated following the method of de Pomerai et al. (35). For quick estimations of protein and nucleic acid concentra- tions, A280:A280 measurements were made.

Isolation of High Molecular Weight Achlya DNA - Mycelia was grown and harvested as previously described then quick frozen in liauid N,. Frozen mvcelia were oowdered with a mortar and pestle. The powdered tissue was mixed with 8 M urea, 0.12 M NaH,PO,, 0.12 M Na,HPO,, and DNA was isolated as described by Dodd et al. (36). Alkaline sucrose sedimentation (37) analysis indicated a molec- ular weight of 4.6 to 6.6 x 105. Small quantities of DNA were isolated from chromatin preparations following the methods de- scribed by Dodd et al. (36).

In Vitro RNA Synthesis-E. coli RNA nolvmerase was diluted with glass-distilled H,O and preincubated with Achlya DNA or Achlya chromatin at either 37” or 0” for 15 min in 200 /*l of preincubation buffer containing 12.5 pmol of Tris/HCl, pH 8.0, 2.5 pmol of dithiothreitol, 2.5 pmol of MgCl,, 20% glycerol, v/v, 50 umol of (NH&SO,. and 100 nmol of K,HPO,. After the preincubation period, RNA’synthesis was started by adding 100 nmoi each of GTP, ATP. CTP. and 10 uCi 13HlUTP (specific activity, 42 Ci/mmol). This’mixture was incubated for another 15 min in-most instances. Where indicated, 10 pg of rifampicin were added along with the nucleoside triphosphate solution. The reaction was terminated by

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8814 Antheridiol Regulation of Transcription in Achlya

the addition of 5% trichloroacetic acid containing 0.01 M sodium pyrophosphate. The resultant precipitate was collected on glass fiber filters (Whatman GF-A) and washed with Hokins solution as previously described (38). The filters were counted in PCS (Amer- sham/Searle) with a Beckman LS-150 scintillation spectrometer.

The commercially prepared E. coli RNA polymerase had a specific activity of 600 units/mg of protein; this corresponds to 50% of the maximum specific activity of 1200 units/mg reported by Burgess (39). These specific activity measurements were used for calculating the amount of enzyme in our assays.

Isolation of in vitro RNA Product - Synthesized RNA was isolated according to the procedure of Tsai et al. (24). Radioactive RNA was precipitated with 50 pg of carrier Achlya RNA in 2.5 volumes of 95% ethanol at -25” for 18 h. The RNA precipitate was collected by centrifugation, washed twice with 95% ethanol, and dried by desic- cation. RNA was suspended in 100 ~1 of sterile glass-distilled H,O and layered on 4.9 ml gradients containing 5 to 20% RNase-free sucrose, 0.01 M NaCl, 0.01 M EDTA, 0.01 M sodium acetate (pH 5.01, and 0.5% sodium lauryl sulfate. The gradients were centrifuged at 45,000 rpm in a Beckman SW 50.1 rotor at 25” for 2.5 h. Achlya ribosomal RNA (40) was used as molecular weight markers to standardize the gradients. Then 25 fractions (0.2 ml each) were collected and the amount of trichloroacetic acid-precipitable radio- activity was measured. The sedimentation coefficient of each frac- tion was determined by reference to the rRNA markers, assuming a linear relationship between position in the gradient and sedimenta- tion coefficient. The chain length of nucleotides (N) was calculated following the procedure described by Tsai et al. (24):

log N = 2.10 log .s*~,~ + 0.644

The number average chain length (N,,,) of the in vitro RNA product was determined by the following equation (37)

N a”g = CinNJLi

where n, is the number of RNA chains and N, is the length of RNA chain in nucleotides.

RESULTS

Preparation and Characterization of Chromatin - When Achlya nuclei are ruptured in a hypotonic solution of Tris/ EDTA, the chromosomal materials is dispersed, but the chro- matin does not form a gel as does the chromatin from most higher eukaryotes. Attempts to isolate Achlya chromatin by the procedures described by Reeder (41) were relatively unsuc- cessful. This procedure, as well as many of the other common chromatin isolation procedures used for higher plant and animal nuclei, depends upon the formation of a translucent swollen gel at low ionic strength. A procedure initially adapted for dinoflagellate chromatin isolation was success- fully used in this study (29). This procedure involves releasing the chromatin by gentle sonication in a 10 mM Tris/HCl, 1 mM EDTA, pH 8.0, medium. After centrifugation to remove nuclear debris, the bulk of the chromatin DNA could be pelleted by adjusting the nuclear lysate to 10 mM CaCl, with solid CaCl,. This procedure recovered 80 to 87% of the total DNA present in the nuclei.

Table I shows the ratio of DNA to RNA to protein of the chromatin and nuclei of A. ambisexualis. The RNA:DNA ratio was 3.3:1 for the isolated nuclei compared to 0.7:1 for the purified chromatin. The DNA to total protein ratio is 1:12 for the isolated nuclei and 1:4.3 for the purified chromatin. DNA to acid-extractable protein values ranged from 1:2.1 in nuclei to 1:1.4 in isolated chromatin.

General Characteristics of Transcription Assay--In vitro transcription of Achlya chromatin by Escherichia coli RNA polymerase followed a linear time course for 30 min and then leveled off. The reactions were dependent on the presence of all four nucleoside triphosphates and added chromatin DNA. The product was sensitive to base hydrolysis and digestion by ribonuclease. Very little endogenous Achlya polymerases

were present in chromatin preparations (see Table IV) (13). Transcription of the chromatin was completely blocked by the addition of 50 pg/ml of actinomycin D.

Initiation Complexes on Achlya DNA-To determine the formation of stable RNA polymerase. DNA initiation com- plexes, increasing amounts of E. coli RNA polymerase were preincubated with Achlya DNA at 0” or 37” for 15 min. The polymerase .DNA complexes were then challenged by adding rifampicin along with ribonucleoside triphosphates and al- lowed to synthesize RNA for an additional 15 min at 37”. Three separate phases of rifampicin-resistant transcription were observed when preincubation of polymerase with DNA was at 37” (Fig. 1). Independent of preincubation temperature, a low level of rifampicin-resistant RNA synthesis was ob- served (Region A, Fig. 1) at low enzyme to DNA ratios. If the RNA polymerase concentration was increased from 1 to 5 pg, a large stimulation of in vitro transcription was observed (Region B, Fig. 1). Initiation complexes in this region were dependent on temperature. No similar RNA chain initiation was apparent when the E. coli polymerase was preincubated with Achlya DNA at 0” (Fig. 1). Increasing the RNA polym- erase concentration from 5 to 20 pg resulted in a further increase in RNA synthesis (Region C, Fig. 11, but at lower rates when compared to Region B.

Tsai et al. (24) using E. coli RNA polymerase and chick DNA defined Regions A, B, and C based upon the expected affinity of E. coli RNA polymerase for DNA. They suggested that Region A represented nicked or single-stranded DNA

TABLE I

Macromolecular composition of Achlya nuclear and chromatin fractions

DNA concentration was determined by the diphenylamine method (34); RNA was estimated following the method of de Pomerai et al. (35); protein concentrations were determined by the method of Lowry et al. (33). Acid-extractable proteins were solubilized from nuclei or chromatin by incubating nuclei in 0.25 M HCl for 60 min at 0°C. Nuclei and chromatin were isolated as described under “Materials and Methods.”

Ratio Fraction

DNA RNA Total protein Acid-extract- able protein

Nuclear 1 3.3 r 0.3 12.0 f 1.1 2.1 * 0.3 Chromatin 1 0.7 f 0.1 4.5 k 0.3 1.4 T 0.07

0 4 6 12 I6 20 RNA Polymerase (89 b

FIG. 1. RNA polymerase saturation curve on Achlya DNA. Esch- erichia coli RNA polymerase (0 to 20 pg) was incubated for 15 min with 2.5 pg of Achlya DNA in the preincubation mixture as de- scribed under “Materials and Methods” at 37” or 0”. Then 50 ~1 of the nucleoside triphosphates and 10 pg of rifampicin were added and the mixture was further incubated for 15 min at 37”. RNA synthesis was terminated and radioactivity determined as described under “Materials and Methods.” 0, preincubated at 37”; 0, preincu- bated at 0”.

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Antheridiol Regulation of Transcription in Achlya 8815

initiation sites and that Region B contained presumptive promoter sites. They based their analyses upon reports that E. coli RNA polymerase binds strongest to nicked or single- stranded regions of DNA (stronger in fact than the true promoter regions of bacteriophage DNA) and reports that RNA polymerase binds to promoter sites more tightly than to random, nonspecific sites (42-44). It was suggested that Re- gion C represented weak binding or nonspecific initiation sites on the DNA (24). RNA polymerase saturation of heat- denatured or sonicated Achlya DNA showed a marked in- crease in the binding capacity and the amount of enzyme needed to saturate Region A (data not shown).

Rifampicin Sensitivity and Temperature Dependence of Initiation of Transcription - Formation of RNA polymerase . DNA initiation complexes were examined to determine tem- perature effects of preincubation in promoting rifampicin- resistant RNA synthesis. Native Achlya DNA, heat-dena- tured DNA, or Achlya chromatin was first incubated with E. coli polymerase for 15 min at either 0” or 37”. After the initial incubation, RNA synthesis was allowed to start upon the addition of either ribonucleotides alone or the ribonucleotides plus rifampicin. In order to minimize the reinitiation of RNA synthesis in the absence of rifampicin, only 2-min reactions were performed at 37”.

The initiation complex formed between RNA polymerase and native DNA at 37” was relatively resistant to rifampicin (Fig. 2A). Preincubation at O”, however, gave a dramatic decrease in the amount of rifampicin-resistant RNA synthesis (Fig. 2A). The initiation complex formed between denatured or single-stranded DNA and RNA polymerase was slightly sensitive to rifampicin yet still showed a temperature depend- ence (Fig. 2B).

Whereas the initiation complex on DNA showed a definite requirement for elevated temperature, the formation of the polymerase preinitiation complex on chromatin did not dem- onstrate a temperature preference (Fig. 20. The chroma- tin.polymerase complex showed enhanced sensitivity to ri- fampicin and a loss of temperature requirement for initiation.

The binding of E. coli polymerase to Achlya chromatin was examined in greater detail and the results are reported in 600 A 600 A

t -flit ’ -flit 7

$ 37%

= 600 Y m

. z

-I E 400

. N x +Rif .-

f 200 3PC

. . -

t I

+Rif . 0°C

f. . -*

5 10

0 C

- Rif

RNA Polymerase lag 1

FIG. 2. Rifampicin (Rif) sensitivity of RNA synthesis on native DNA, denatured-DNA, and chromatin. The reactions were per- formed following the procedure described in Fig. 1 with the excep- tions of carrying out one set of experiments in the absence of rifampicin and all reactions were incubated for only 2 min. A, 2.5 pg of native Achlya DNA. B, 2.5 pg of heat-denatured Achlya DNA. C, 15 pg of Achlya chromatin isolated from non-hormone- treated cultures of Achlya. 0, preincubated at 37” and incubated without rifampicin; W, preincubated at 37” and incubated with 10 pg of rifampicin; A, preincubated at 0” and incubated with 10 pg of rifampicin.

Fig. 3. As in the case of E. coli polymerase and chick oviduct chromatin (24), the enzyme saturation curve was viewed as having two phases. Firstly, there was a steep increase in RNA synthesis after adding increasing quantities of polymer- ase and secondly, this was followed by a very gradual enhance- ment of nucleoside monophosphate incorporation with in- creased polymerase concentration (Fig. 3). The small increase in the amount of RNA transcribed in the second phase has been suggested to result from initiation at weak affinity or nonspecific sites, which are perhaps uncovered during addi- tion of large quantities of enzyme protein (24). The transition points between these two phases were very reproducible for a given chromatin. The coordinates of the transition point in the saturation curve were characteristically different when chromatin was isolated from Achlya induced for specific time periods with antheridiol and are qualitatively proportional to template activity (see Fig. 6).

Determination of RNA Chain Sizes-In order to quantitate RNA chain initiation sites on DNA and chromatin, the size of the RNA products synthesized in vitro were measured. The conditions for preincubation, RNA synthesis, termination of synthesis, product purification, and analysis were identical with those described by Tsai et al. (24). In order to determine if ribonuclease was a contaminant of our chromatin prepara- tions, purified radioactive total cellular RNA was incubated with the chromatin-transcription mixture lacking the labeled UTP. After incubation for 15 min at 37”, the chromatin fraction was pelleted by centrifugation. Sucrose gradient anal- ysis of the supernatant fractions are shown in Fig. 4C. The profile showed very little degradation of the ribosomal 26 S and 18 S RNA species. Unlike the chicken oviduct system which requires heparin to inhibit chromatin ribonucleases (24), heparin was not required for the Achlya in vitro tran- scription system (Fig. 40. Furthermore, when heparin was added to the in vitro transcription mixtures, it had a slight inhibitory effect on the rate of incorporation.

RNA was synthesized under conditions where rifampicin was added along with the nucleoside triphosphates. The reactions were allowed to incubate for 30 s, 3 min, and 15 min. The RNA product was isolated and fractionated on sucrose gradients. Fig. 4A indicates an increase in RNA chain size during 15 min of synthesis. The number average size of RNA chains was calculated to be -575 nucleotides for 15-min incubations. The size distribution was similar for all

measured Achlya chromatin preparations (isolated at 1 and 4 h after hormone treatment) and was considerably larger than

z P 8 600-

B .E

-.2.vp*.-. -.____ -.-.-.-

0 10 20 30 RNA Polymsrass ((IS 1

FIG. 3. Temperature dependence on the formation of rifampicin- resistant RNA polymerase . chromatin complexes. Procedures were the same as those described in Fig. 1 with 25 Fg ofAchlya chromatin from non-hormone-treated tissues. 0, preincubated at 37”; 0, prein- cubated at 0”; A, RNA polymerase preincubated alone without any Achlya chromatin at 37”.

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8816 Antheridiol Regulation of Transcription in Achlya

B 16 24 32 40

s Value

FIG. 4. Sucrose gradient analysis of RNA synthesized on Achlya chromatin and DNA. A, Achlya chromatin (50 pg from 4 h antheri- diol-treated cultures) was incubated with 60 pg of Escherichia coli RNA polymerase for 15 min at 37”. RNA synthesis was started by the addition of nucleoside triphosphate and incubated for 15 min (. . .O. . .) or with the addition of nucleoside triphosphate and 40 pg/ml of rifampicin and synthesized for 30 s (- 0 - ), 3 min (W, or 15 min (A). RNA was purified and the S value determined on sucrose gradients as described under “Materials and Methods.” B, Achlya DNA (15 pg) was incubated with 60 pg of E. coli polymerase and the reaction was carried out at 37” in the presence of 40 pg/ml of rifampicin and then following the procedures described for A. C, purified Achlya total cellular RNA (14,000 cpm/pg; 0) was incubated with the transcription assay minus radioactive nucleotides for 15 min at 37” (W) and with 800 pg/ml of heparin (-. -). Chromatin was pelleted (3000 x g for 10 min) after making the reaction mixture 10 rnM with solid CaCl, and the supernatant was immedi- ately applied to sucrose gradients.

the RNA synthesized from native Achlya DNA (Fig. 4R) (Table II).

Utilizing curves representing RNA polymerase saturation ofAchlya chromatin (Fig. 6), the coordinates of the transition point were used to determine the quantity of RNA polymerase molecules bound to high affinity sites and the quantity of RNA chains initiated. If it is assumed that the E. coli polymerase binds to chromatin in its monomeric form (480,000), the number of binding sites can be calculated from the number of molecules of RNA polymerase required to reach the transition point (24):

Number of binding sites = E x 0.5 x N pg of DNA 4.8 x lo5 x DNA

where E = amount of RNA polymerase bound in grams, N = Avogadro’s number 6.02 x 10Z3, and DNA = amount of DNA or chromatin DNA used in picograms. The quantity of rifam- picin-resistant initiation sites can be calculated from the size and the total amount of RNA synthesized and the specific activity of the radioactive nucleotide (315 cpm/pmol).

Number of initiation sites cpm x 1Om’2 x 4 x N pg of DNA 315 x DNA x size

where cpm = cpm of UMP incorporated at the transition

TABLE II

RNA polymerase binding sites and initiation sites on Achyla DNA and chromatin Polymer- ase mole- Size of RNA

Cd% RNA syn- chain ini- ‘$~$~~~

(binding thesized tiation ZYlTll2 sites”/ g

x (nucleo- tides) bound

ofDN )

DNA Region A” Region B”

Achyla chromatin 0 h (no hormone)’ 1 h hormone’ 4 h hormone’

x10-5 x10 4 7"

2.5 320 5.7 22.3 12.5 320 86.0 68.8

5.1 575 0.8 1.5 8.2 575 1.7 2.0

10.0 575 2.3 2.3

a Binding sites and initiation sites were calculated as described under “Results” after the procedure of Tsai et al. (24).

b Data calculated from Fig. 1. c Data calculated from Fig. 6.

point, and size = number of average chain length of RNA (24). For these calculations, it was assumed that UMP incor- porated was equal to 25% of the total nucleotides incorporated and that the RNA polymerase was 50% active (see “Materials and Methods”).

The number of enzyme binding sites and initiation siteslpg of DNA were calculated and are reported in Table II. Region A showed very few initiation sites/enzyme bound, whereas Region B showed a high correlation between the number of binding sites and the number of RNA chain initiations. The efficiency of initiation was also greater, since 69% of the polymerase molecules added to DNA within Region B were successful in initiating RNA chains, whereas only 22% of the polymerase bound was successful in Region A.

In the case of chromatin, we determined that only 50 to 60% of the E. coli RNA polymerase added to the incubation reaction ever bound to the chromatin. We made this estima- tion by incubating a normal reaction mixture and removing the chromatin-bound polymerase by centrifugation. We then assayed the supernatant fractions against pure DNA under standardized conditions and determined the unbound residual RNA polymerase. Optimal binding of polymerase to chroma- tin required at least a lo-min preincubation step. Taking this into consideration, the calculated values for Achlya chromatin were 1 polymerase binding site/l766 nucleotide pairs for O-h chromatin, 1 polymerase binding site/1073 nucleotide pairs for l-h hormone chromatin, and 1 polymerase binding site/ 880 nucleotide pairs for 4-h hormone chromatin. One initiation site/6 x lo4 nucleotide pairs was determined for O-h chromatin, 1 initiation site/2.5 x lo4 nucleotide pairs for l-h hormone chromatin, and 1 initiation site/2.15 x lo4 nucleotide pairs for 4-h hormone chromatin. These values corresponded to 2.5 x lo4 binding sites and 730 rifampicin-resistant initiation sites for O-h chromatin/haploid genome ofAchZya (4.4 x lo7 nucleo- tide pairslhaploid genome) (36). For chromatin isolated from l-h hormone-treated tissue the values were 2.5 x 10” binding sites and 1760 rifampicin-resistant initiation sites; for chro- matin isolated from 4-h hormone-treated cultures, the values were 2.6 x 10“ binding sites and 2046 initiation siteslhaploid Achlya genome. Our estimations of the number of initiation sites found on Achlya chromatin was considerably fewer than reported for chicken (24) but very similar to the 2300 number of diverse RNA species reported to exist in growing cultures of Achlya (45). Although the number of measured binding sites is similar to the number of different gene transcripts

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Antheridiol Regulation of Transcripption in Achlya 8817

reported to exist in Achlya, we have no evidence to suggest that the initiation sites measured in vitro are identical with those operative in vivo.

Unlike the process of initiation on Achlya DNA, the effl- ciency of initiation on Achlya chromatin was less than 3% (Table II). This extremely low efficiency may be explained either by suggesting that most of the polymerase binds to “storage regions” on the chromatin, perhaps adjacent to true initiation sites or that the preinitiation complex between chromatin and enzyme may be extremely sensitive to rifam- picin (24). Tsai et al. (24) demonstrate the existence of such “storage regions” in chicken oviduct chromatin.

The rates of in vitro RNA chain propagation for chromatin from control, l-h antheridiol-treated, and 4-h antheridiol- treated Achlya cultures were estimated by first adding E. coli RNA polymerase to chromatin under conditions previously described. This resulted in the formation of the RNA polym- erase. DNA complex. This mixture was incubated for 15 min at 37” and then RNA chain initiation and elongation was determined by the addition of the nucleoside triphosphates and rifampicin. After 05, 3-, or 15-min incubation periods, the rate of RNA chain elongation was estimated by dividing the number average molecular weight of the synthesized chains by the total time of synthesis (24). No differences were observed in the rates of synthesis between the various chro- matins. The initial rate of chain elongation was five nucleo- tides/s at 30 s and was reduced to three and one-half nucleo- tides/s at 3 min and two nucleotidesls at 15 min. The slower rates of chain growth at the later times has been explained on the basis of chain termination (24). Chain elongation rates on purified DNA were similar to those measured on chroma- tin.

Effects of Antheridiol on Chromatin Template Capacity- Chromatin template activity was determined under conditions where radioactive UTP was incorporated into RNA at saturat- ing levels of E. coli RNA polymerase and nucleotides (15, 16). Increasing concentrations of Achlya chromatin were preincu- bated with RNA polymerase for 15 min at 37”. Total RNA synthesis was determined 15 min after nucleotide addition. Fig. 5 demonstrates that the incorporation of nucleotides into an RNA product was proportional to the template concentra- tion. Following these methods, template activity was mea- sured during antheridiol-induced formation of male sex organ initials. At 1 h after hormone t,reatment there is a 7.7% increase in template activity which increases to 31%, 4 h after hormone addition, when the morphological appearance of the male sex organ initial becomes apparent (Fig. 5; Table III). These data suggest that during the antheridiol-induced differentiation of male sex organ initials, there was a corre- sponding quantitative increase in gene transcription.

Table III shows that the template capacity (percentage of total RNA synthesis on chromatin in comparison to pure DNA as a template) of Achlya chromatin was extremely high. Values of 49% for control (0 h), 52% for l-h hormone chromatin, and 64% for 4-h hormone chromatin were calcu- lated.

Titration of Achlya Chromatin with E. coli RNA Polymer- use - Fixed concentrations (25 pg of chromatin DNA) of Ach- lya chromatin were titrated with increasing concentrations of E. coli RNA polymerase and preincubated together for 15 min at 37”. RNA synthesis was initiated by the addition of the nucleoside triphosphates and rifampicin. The total reac- tion mixture was allowed to synthesize RNA. With increasing amounts of the bacterial polymerase, a transition point was

reached. This transition point represents the number of RNA polymerase molecules needed to saturate high affinity initia- tion sites on the chromatin under conditions of drug inhibition of RNA chain reinitiation (Fig. 6). At 1 and 4 h after the addition of antheridiol, a greater quantity of RNA polymerase was required to saturate the Achlya chromatin prepared from hormone-treated cultures (Fig. 6). The amount of polymerase needed to saturate chromatin is representative of the number of RNA polymerase binding sites available on the chromatin (Table II) (24). Within 1 h after the administration of antheri- diol to untreated cultures the amount of RNA polymerase needed to saturate the chromatin is increased from 8 pg/25 pg of chromatin DNA in control (non-hormone-treated) chro- matin to 13 pg/25 pg of chromatin DNA. At 4 h a further increase (16 pg/25 pg of chromatin DNA) was measured.

Tsai et al. reported that rifampicin-resistant initiation sites on chromatin have certain properties similar to those seen on single-stranded or nicked DNA (24). It is therefore possible that fragmentation of chromatin DNA may be a mechanism for increasing the number of RNA initiation sites. DNA isolated separately from unstimulated and antheridiol-simu- lated chromatin and then exposed to polymerase titration assays demonstrated titration curves that were identical with those shown in Fig. 1. The level of initiation due to Region A

Lk = 800 - F s-7 U ; 600 - E ; 400 - .E

z 200 - E P

Chromatin DNA ( pti ) FIG. 5. Concentration dependence of the template activity of

Achlya chromatin. Achlya chromatin from O-h (O), l-h (A,), and 4-h (m) antheridiol-treated cultures was incubated with 22 pg of Esche- richia coli RNA polymerase. Nucleoside triphosphates were added and the reaction incubated for 15 min at 37”. The reactions were terminated as previously described and the results are expressed as picomoles of 13H]UMP incorporated/reaction mixture. No incorpora- tion of labeled UMP into RNA was observed at zero concentration of chromatin DNA.

TABLE III

Effect of antheridiol on Achlya chromatin activity

Achlya chromatin (25 pg of DNA) or DNA (2.5 pg) and 22 pg of RNA polymerase were allowed to synthesize RNA under conditions described under “Materials and Methods.”

Source of template

- PHIUMP

Time of incorpora- hCWZ3Se

hormone tion into over un- Template treatment R;$y

stimulated capacity” chromatin

h pm01 % Achlya chromatin 0 13 49

1 14 7.7 52 4 17 31.0 64

Achlya DNA 26.5 100

fl The percentage of total RNA synthesized on chromatin in comparison to pure DNA as a template.

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8818 Antheridiol Regulation of Transcription in Achlya

of the titration curve was similar for DNA isolated from 0 h (control) and DNA isolated from chromatin 4 h after hormone treatment. The differential amount of RNA polymerase re- quired to reach the transition point in chromatin isolated from antheridiol-treated tissue does not appear to be related to fragmentation of DNA.

Endogenous Polymerase Activity of Chromatin - The num- ber of initiation sites could be greatly misinterpreted if high levels of endogenous RNA polymerase were present on the Achlya chromatin. It has been suggested that as much as one-tenth of the total RNA polymerase II in tissue can be present in purified chromatin preparations (46). We therefore examined the levels of endogenous Achlya RNA polymerases in 25 pg of chromatin DNA samples. Our assay conditions revealed that less than 0.5% of the total RNA synthesized was due to the endogenous Achlya polymerases. It has also been suggested that incubation of E. coli RNA polymerase with chromatin may enhance the reaction of endogenous

c 4 hours 30

RNA Polymerase (,pg 1

FIG. 6. Titration of Achlya chromatin with Escherichia coli RNA polymerase. Increasing amounts of RNA polymerase were preincu- bated with (25 pg of chromatin DNA) of Achlya chromatin isolated from antheridiol-treated and control cultures for 15 min at 37”. RNA synthesis was started with the addition of the four nucleoside triphosphates and 10 pg of rifampicin. Samples were assayed for the incorporation of FHlUMP into trichoroacetic acid-precipitable material.

TABLE IV

Effect of antheridiol on endogenous RNA polymerme activity of Achlya ambisexualis chromatin

Achlya chromatin (25 pg of chromatin DNA) was preincubated in the presence or absence of Escherichia coli RNA polymerase (50 pg) for 15 min at 37”. The polymerase reaction was started by the addition of the four nucleoside triphosphates. Column A, chromatin was preincubated without any exogenous E. coli polymerase. Col- umn B, chromatin was preincubated with E. coli RNA polymerase. Column C, chromatin was preincubated with E. coli polymerase and 10 fig of a-amanitin.

Antheridiol treat- merit

[3H]UMP incorporated into Incorpora- RNA tion by en-

dogenous A B C enzvmes

pm01 % 0 h (control) 2.9 1215 1185 co.5 lh 5.3 2240 2215 <0.5 4h 6.7 2730 2745 CO.5

chromatin-bound RNA polymerase. When up to 10 pg of a- amanitin were added to reaction mixtures containing Achlya chromatin and E. coli polymerase, there was no measurable effect on the level of rifampicin-resistant RNA synthesis from control or antheridiol-treated Achlya chromatin (Table IV).

DISCUSSION

The characteristics of the chromatin isolated from the Oomycete fungus Achlya ambisexualis were similar to the characteristics of chromatin isolated from several other lower eukaryotes. Considerable variation exists in the literature concerning the RNA:DNA ratios of fungal chromatin. Values as high as 3.8:1 (for Saccharomyces) to as low as 0.05:1 (for

Neurospora, Phycomyces, and Microsporum) have been re- ported (4’7). The value of 0.7 obtained for A. ambisexualis is similar to values obtained for some higher eukaryotes and to values recently reported for several true fungi (47-49). The value of 4.5:1 for total chromosomal protein:DNA ratio is higher than some ratios reported (48, 49) but similar to others (50, 51). Horgen et al. (31) reported the existence of histone- like basic chromosomal proteins in Achlyu nuclei. The ratio of acid-extractable protein:DNA in A. ambisexualis is similar to the values reported for Aspergillus and Sacchuromyces which is comparable to values reported for other eukaryotes

(49, 50). Furthermore, Achlya possesses a typical nucleosome chromatin subunit structure.’

Chromatin from Achlya was capable of being transcribed by heterologous Escherichiu coli RNA polymerase. In vitro

transcription was dependent upon the presence of added chromatin DNA, the four nucleoside triphosphates, and was inhibited by the addition of actinomycin D. The in vitro assayed followed normal kinetics of incorporation and the product synthesized was established as being RNA.

In complex animal systems steroids appear to affect gene transcription in the target cells. Chromatin template activity is enhanced in rat muscle after testosterone treatment (23), in rat liver after hydrocortisone administration (221, in ham- ster uterine tissue during the estrous cycle (52) and in chicken oviduct after estrogen treatment (25). Teng and Hamilton

(53) have reported the stimulation of template capacity of rat uterine chromatin following the injection of estrogens to ovariectomized animals. Horgen (13) has shown that incuba- tion of chromatin isolated from non-hormone-treated cultures of Achlya with antheridiol and cytosol from male strain E87 dramatically increased the RNA synthetic capacity of the chromatin.

In this study, chromatin template activity was used as a means for assessing changes in transcription after the addition of the sex hormone antheridiol to male strains of the aquatic fungus A. ambisexuulis. The process of RNA chain initiation has been separated from other transcriptional events by titrating Achlya DNA or chromatin with increasing amounts of E. coli RNA polymerase in the presence of the drug rifampicin. The use of heterologous bacterial RNA polymerase to transcribe eukaryotic chromatin has been a controversial experimental method. Some authors report that bacterial polymerases do not transcribe eukaryotic chromatin accu- rately (41, 54) and indeed may even transcribe repressed DNA sequences. Recently, Biessmann et al. (55) have reported that E. coli RNA polymerase does not transcribe DNA with a high degree of fidelity but does transcribe sequences of the chromatin which are representative of the in vivo RNA

1 J. C. Silver, personal communication.

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Antheridiol Regulation of Transcription in Achlya 8819

frequency distribution. Tsai et al. (26) have demonstrated convincingly that E. coli RNA polymerase and RNA polymer- ase II from hen oviduct compete for the same initiation sites on oviduct chromatin. In contrast to chromatin, however, the majority of the initiation sites on naked DNA which are utilized by the hen polymerase are different from those uti- lized by E. coli holoenzyme. Based on the procedures devel- oped by Tsai et al. (24) and the polymerase competition experiments described above (26), we were able to saturate and quantify the RNA polymerase binding and initiation sites on Achlya DNA and chromatin.

The binding of RNA polymerase and the formation of initiation complexes were studied using Achlya DNA. At lower polymerase concentrations, the enzyme binds nonspecif- ically to single-stranded or nicked regions (Fig. 1, Region A). Temperature had no effect on the formation of initiation complexes in this region. Tsai et al. reported that enzymatic and physical damage of the DNA increased the amount of RNA polymerase needed to saturate this region. If heat- denatured or sonicated Achlya DNA was used rather than the larger molecular weight DNA (4 to 6 x 105), more RNA polymerase was required to saturate Region A (data not shown). It has been reported that when RNA polymerase binds to Region A, the enzyme does not undergo a conforma- tional change which is required for the stable initiation complex. With higher RNA polymerase levels, temperature- dependent initiation complexes form on Region B of the curve (Figs. 1 and 2, A and B) (24). The use of single-stranded DNA eliminated Region B (Fig. 2B). The polymerase that binds to the DNA in Region B has been suggested to be similar to the binding of polymerase to strong promoter sites found on phage DNA (56, 5’7). With increasing concentrations of E. coli polymerase a third region (Region C, Fig. 1) is observed and is thought to be due to the enzyme binding to weak or secondary RNA initiation sites.

The RNA polymerase saturation curve for isolated Achlya chromatin indicated only one major class of initiation sites which were temperature-independent. Tsai et al. (24) reported similar results and suggested that initiation of RNA synthesis by this complex resembled initiation on nicked or single- stranded DNA. Since initiation on DNA which was purified from chromatin isolated at different times after hormone treatment was similar, the differences observed are not due to nicked chromatin DNA. The number of initiation sites on isolated chromatin is positively correlated to the synthetic functions of the cell. Chicken erythrocytes, known to have a low number of expressed genes (581, also had few RNA polymerase binding and initiation sites on its chromatin (24). The temperature requirement for the formation of rifampicin- resistant preinitiation complexes on purified DNA has been interpreted as a result of an energy requirement for strand separation or unwinding over short local regions at the initia- tion sites (24). The lack of temperature dependence for initia- tion of RNA synthesis on chromatin may be a result of the interaction of DNA with its chromosomal proteins (24). The observation that initiation of RNA synthesis on chromatin is more sensitive to rifampicin than initiation of RNA synthesis on DNA has been explained on the basis of a model proposed for the promoter region of T, phage and has been thoroughly discussed elsewhere (21, 24).

Within 1 h after the addition of antheridiol to vegetatively growing cultures of Achlya, a 7.7% increase was detected in the activity of the isolated chromatin (Table III). The level increased to 31%, 4 h after the addition of antheridiol. The

template capacity values calculated for in vitro transcription of Achlya DNA (49 to 64%, Table III) were considerably higher than the 2.0 to 9.0% template capacities reported for diethylstilbestrol-stimulated chicken oviduct chromatin (25). During the developmental cycle of another eukaryotic mi- crobe, Dictyostelium discoideum, it has been reported that over 50% of the single copy DNA is transcribed (59). Because the Achlya genome is 25 times smaller than the genome of chicken, one might predict that a larger percentage of the DNA would be expressed. Furthermore, RNA excess, DNA- RNA hybridization studies with Achlya mRNA and unique DNA indicate that 40 to 60% of the unique sequences of the Achlya genome are expressed during antheridiol-induced sex- ual differentiation.2 These observations taken all together strongly suggest that a large percentage of the total genetic information present in the nucleus of Achlyu is expressed during growth and an even larger percentage is expressed during steroid-mediated differentiation of the male sex organ.

The results of this study show that the steroid sex hormone antheridiol regulates transcription in male strains of Achlya by increasing the number of RNA polymerase initiation sites on nuclear chromatin. Whereas the level of RNA chain initiations on Achlyu chromatin increased after antheridiol administration to vegetatively growing cultures, the RNA chain propagation rate and the size of the average RNA product synthesized remained unchanged. This observation while being reproducible for the in vitro transcriptional con- ditions employed may not necessarily hold true in uiuo. In complex animal systems, it has been suggested that steroid hormone action involves interaction of steroid.receptor com- plexes with acceptor sites on chromatin (14). This interaction then results in increasing the number of available initiation sequences in the DNA template and the synthesis of new RNA sequences (60). These hypotheses are supported by evidence that shows a direct correlation in the number of nuclear-bound steroid receptor molecules and polymerase ini- tiation sites (61). The steroid. hormone. receptor complex may influence transcription by causing a structural change in the chromatin allowing for exposure of previously unavailable polymerase binding and initiation sites. Once these sites are available, the synthesis of steroid-induced gene transcripts will soon follow. These observations all suggest transcrip- tional control as the primary mechanism of steroid hormone action in complex animal systems.

Compared to animal systems, relatively little is known about the complex mechanisms operative in simple eukaryotes that respond to steroid hormones. The information that is available suggests that the aquatic fungus Achlyu responds to steroid hormones in a manner similar to much more complex eukaryotes (2). There is “target-tissue” specificity associated with hormone interactions in Achlya and evidence suggests that hormone-cytosol interactions similar to the hormone-receptor mechanisms operable in complex animal systems also exist (2, 13). The results of this study also suggest some direct analogies to more complex systems. It appears that transcriptional control mechanisms are operable during antheridiol-induced sexual differentiation in Achlya.

Achlya is perhaps the most primitive eukaryotic organism to produce and respond developmentally to steroid sex hor- mones. Because Achlya is a microorganism, because it is extremely fast growing, because the entire male thallus acts as target tissue for antheridiol, and because it is possible to generate mutants of Achlya for genetic experiments (some-

2 N. A. Straus and P. A. Horgen, manuscript in preparation.

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8820 Antheridiol Regulation of Transcription in Achlya

thing not so easily done with the target tissue of adult (1976) J. Biol. Chem. 251, 1960-1968

animals), Achlya has the potential to lend itself as a useful 27. Griffin, D. H., and Breuker, C. (1969) J. Bacterial. 98, 689-696

system with which to examine the complex control mecha- 28. Barksdale, A. W., and Lasure, L. (1973) Bull. Torrey Bot. Club

nisms associated with steroid hormone mediation of cellular 100, 199-202

29. Rizzo, P. J., and Nooden, L. D. (1974) Biochim. Biophys. Acta differentiation. 349, 402-414

Acknowledgments -The technical assistance of E. Thomp- 30. Hnilica, L. (1972) The Structure and Biological Function of

son is gratefully acknowledged. Antheridiol was a generous Histones, p. 137, Chemical Rubber Company, Cleveland, Ohio

31. Horgen, P. A., Nagao, R. J., Chia, L. S. Y., and Key, J. L. gift of Dr. Trevor McMorris, University of California, La (1973) Arch. Microbial. 94, 249-258 Jolla. 32. Panyim, S., and Chalkley, R. (1969) Arch. Biochem. Biophys.

1. 2.

3. 4. 5. 6.

7.

8. 9.

10.

11.

12. 13.

14. 15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

REFERENCES

Barksdale. A. W. (1969) Science 166. 831-837

130, 337-346 33. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R.

J. (1951) J. Biol. Chem. 193, 265-275 Horgen, P: A. (1977) in Eukaryotic Microbes as Model Deuelop- 34. Giles, K. W., and Myers, A. (1965) Nature 206, 93

mental Systems (O’Dav. D. H.. and Horgen, P. A., eds) pp. 35. de Pomerai, P. I., Chesterton, C. J., and Butterworth, P. H. W. 272-294, “Marcel Dekker; New York -

__ (1974) Eur. J. Biochem. 46, 461-471

Thomas, D., and Mullins, J. T. (1967) Science 156, 84-85 36. Dodd, J. G., Horgen, P. A., and Straus, N. A. (1975) Cytobios Silver. J. C.. and Horeen. P. A. (1974) Nature 249. 252-254 13. 31-36 Timberlake, W. E. (1976) Deu. BibZ. 5i, 202-214 ’ 37. Studier, S. W. (1965) J. Mol. Biol. 11, 373-390 Groner, B., Hynes, N., Sippel, A., and Schatz, G. (1976) Nature 38. Horgen, P. A., and Key, J. L. (1973) Biochim. Biophys. Acta

261, 599-601 294,227-235 Horgen, P. A., Smith, R., Silver, J. C., and Craig, G. (1975) 39. Burgess, R. R. (1969) J. Biol. Chem. 244, 6160-6167

Can. J. Biochem. 53, 1341-1345 40. Jaworski, A. J., and Horgen, P. A. (1973) Arch. Biochem. Horgen, P. A., and Ball, S. F. (1974) Cytobios 10, 181-185 Biophys. 157, 260-267 Thomas. D.. and Mullins. J. T. (1969) Phvsiol. PZant. 22. 347- 41. Reeder. R. H. (1973) J. Mol. Biol. 80, 229-241

353 ’ ’ Mullins, J. T., and Ellis, E. A. (1974)Proc. Natl. Acad. Sci. U.

S. A. 71, 1347-1350 Griffin, D. H., Timberlake, W. E., and Cheny, J. C. (1974) J.

Gen. Microbial. 80, 381-388 Lasure, L., and Griffin, D. H. (1974) Mycologia 66, 391-396 Horgen, P. A. (1977) Biochem. Biophys. Res. Commun. 75,

1022-1027 O’Malley, B. W., and Means, A. R. (1974) Science 183, 610-620 O’Malley, B. W., and McGuire, W. L. (1968) Proc. Natl. Acad.

Sci. U. S. A. 60, 1527-1534 Means. A. R.. Comstock. J. P.. Rosenfeld. G. C.. and O’Mallev.

42. Hinkle; D. C., and Chamberlin, M. (1970) Cold Spring Harbor Syrup. Quant. Biol. 35, 65-72

43. Hinkle, D. C., and Chamberlin, M. J. (1972) J. Mol. Biol. 70, 157-185

44. Hinkle, D. C., Ring, J., and Chamberlin, M. J. (1972) J. Mol. Biol. 70, 197-207

45. Timberlake, W. E., Shumard, D. S., and Goldberg, R. B. (1977) Cell 10. 623-632

46. Cox, R. $. (1973) Eur. J. Biochem. 39, 49-61 47. Leighton, T., Leighton, F., Dill, B., and Stock, J. (1976)

Biochim. Biophys. Acta 432, 381-394 48. Hsiana. M. W., and Cole, R. D. (1973)J. Biol. Chem. 218, 2007-

B. W. (1972)&oc. Nail. AC&. Sci. U. s. A. SS, 1146-1150 “’ 2013’ Harris. S. E.. Rosen. J. M.. Means. A. R.. and G’Mallev. B. W. 49. Felden. R. A., Sanders, M. M., and Morris, N. R. (1976) J. Cell

(1975) Biochemistry 14, 2672-2081 “I

Bio1..68, 430-439 O’Malley, B. W., McGuire, W., Kohler, P., and Korennan, S. 50. Wintersberger, U., Smith, P., and Letnansky, K. (1973) Eur. J.

(1969) Recent Prog. Horm. Res. 25, 105-160 Biochem. 33, 123-130 Cox, R., Haines, M., and Carey, N. (1973) Eur. J. Biochem. 32, 51. Goff, G. C. (1976) J. Biol. Chem. 251, 4131-4138

513-524 52. Warren, J. C., and Barker, K. L. (1967) Biochim. Biophys. Acta Spelsberg, T. C., Mitchell, W. M., Chytil, F., Wilson, E. M., 138, 421-423

and O’Malley, B. W. (1973) Biochim. Biophys. Acta 312, 765- 53. Teng, C. S., and Hamilton, T. H. (1968) Proc. N&l. Acad. Sci. 778 U. S. A. 60, 1410-1417

Towle, H. C., Tsai, M. J., Hirose, M., Tsai, S. Y., Schwartz, R. 54. Chesterton, C. J., and Butterworth, P. (1971) Eur. J. Biochem. J., Parker, M. G., and O’Malley, B. W. (1976) in Molecular 25, 463-470 Biology of Hormone Action (Papaconstantinou, J., ed) pp. 55. Biessmann, H., Gjerset, R. A., Levy, B., and McCarthy, B. I. 107-136, Academic Press, New York (1976) Biochemistry 15, 4356-4363

Dahmus, M. E., and Bonner, J. (1965) Proc. N&Z. Acad. Sci. 56. Bantz, E. K. F., and Bantz, F. A. (1970) Nature 226, 1219-1222 U. S. A. 54, 1370-1375 57. Chamberlin, M. J., and Ring, J. (1972) J. Mol. Biol. 70,221-237

Breuer, C. B., and Florini, J. R. (1966) Biochemistry 5, 3857- 58. Siligy, V., and Neelin, J. (1970) Biochim. Biophys. Acta 213, 3865 380-390

Tsai, M. J., Schwartz, R. J., Tsai, S. Y., and O’Malley, B. W. 59. Firtel, R. A. (1972) J. Mol. Biol. 66, 363-377 (1975) J. Biol. Chem. 250, 5165-5174 60. Liarakos, C. D., Rosen, J. M., and O’Malley, B. W. (1973)

Schwartz, R. J., Tsai, M. J., Tsai, S. Y., and O’Malley, B. W. Biochemistry 12, 2809-2816 (1975) J. Biol. Chem. 250, 5175-5182 61. Buller, R. E., Schwartz, R. J., Schrader, W. T., and G’Malley,

Tsai, M. J., Towle, H. C., Harris, S. E., and O’Malley, B. W. B. W. (1976) J. Biol. Chem. 251, 5178-5186

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R B Sutherland and P A Horgenthe simple eukaryote, Achlya ambisexualis.

Effects of the steroid sex hormone, antheridiol, on the initiation of RNA synthesis in

1977, 252:8812-8820.J. Biol. Chem. 

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