Proc. Natl. Acad. Sci. USAVol. 83, pp. 8839-8843, December 1986Biochemistry

Nuclease resistance and the enrichment of native nuclear acceptorsites for the avian oviduct progesterone receptor

(DNase I/Southern-blot analyses/Scatchard analyses/nuclear matrix/purified receptor I)

JIM HoRA, MICHAEL J. HORTON, DAVID 0. TOFT, AND THOMAS C. SPELSBERG*

Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905

Communicated by Ralph T. Holman, August 4, 1986

ABSTRACT High-affinity nucleoprotein acceptor sites forthe avian oviduct progesterone receptor (PR) have been en-riched by a combination of nuclease digestion and centrifuga-tion. These enriched binding elements exhibited markedlyenhanced PR binding on a per mass DNA basis compared tochromatin (20- to 25-fold) or dehistonized chromatin (4- to5-fold). Electrophoretic analysis of the nuclease-resistant DNAshowed that there is a set of DNA fragments of 100-150 basepairs that are protected from digestion. Excessive digestionresulted in smaller DNA fragments and a loss of PR bindingactivity. The PR binding was saturable using a crude receptorpreparation and displayed a competition with the same recep-tor preparation that was labeled with nonradioactive proges-terone. The enhanced binding was also demonstrable usinghighly purified receptor preparations that exhibit two classes ofbinding sites both of which are of high affinity and saturable asassessed by Scatchard analyses. These two high-affinity classesof binding sites are shown to be competed by unlabeled purifiedPR. The nuclease resistance of these nucleoprotein acceptorsites from chromatin is a property similar to the nuclear matrixbinding sites suggesting a relationship between these two classesof nuclear acceptor sites.

Based on the presently accepted mechanism of steroidhormone action, the interaction of a steroid receptor-hor-mone complex with the nucleus is an important step in theinduction of specific gene products. A great deal of effort hasbeen invested by a number of laboratories in trying todetermine the relevant receptor-nuclear interactions. De-spite these efforts, the exact nature of the nuclear bindingsites (acceptor sites) is not completely understood. Steroidreceptors have been shown to bind to DNA (1), RNA (2),chromatin (3), and ribonucleoprotein particles (4).

This laboratory has been concentrating on the specificinteraction ofthe chicken oviduct progesterone receptor (PR)with nuclear acceptor sites in oviduct chromatin. In contrastto the receptor binding to pure DNA sequences alone (5, 6),the binding of the oviduct PR to subfractions of chromatincontaining nonhistone protein-DNA complexes, termednucleoacidic protein or NAP, has been shown to be receptordependent and saturable, of high affinity (7-11), to bereceptor specific (12), and to generate patterns of bindingssimilar to that measured in vivo (13-16). Further, the capacityof the PR to bind to nuclear acceptor sites reflects the abilityof progesterone to alter RNA polymerase activity in nuclearrun-off experiments (17, t) and to specifically induce theavidin gene (18). A specific subset of nonhistone proteins(acceptor proteins) has been shown to be necessary for thegeneration of specific nucleoprotein acceptor sites (10, 11,19-21).

In other organisms, similar nuclear acceptor sites com-posed of tightly bound (to DNA) nonhistone proteins havebeen reported for the estrogen receptor/chicken oviductsystem (17), for systems involving the estrogen and proges-terone receptors in sheep brain (22), in hamster uteri (t), incow, rabbit, and human uteri (21, 23, 24), for systemsinvolving the glucocorticoid receptor in both rat liver (25) andhuman leukemic cell line system (26), and finally for theandrogen receptor rat prostate system (27). Thus, the accep-tor protein-DNA complex as a potential nuclear acceptor sitemodel for steroid receptors seems to be universal. In thisreport, evidence is presented that these complexes areresistant to nuclease digestion, generating small nucleopro-tein fragments that maintain the specific PR binding similar tothat found in whole intact chromatin.

MATERIALS AND METHODS

Isolation of Partially Purified and Purified Avian OviductPR. The procedures for the isolation, labeling, and partialpurification of PR from estrogen-stimulated chicken oviductused in this study were modifications of methods described(16). Highly purified PR was isolated by the method of Puriand Toft (28) in the presence of 10 mM sodium molybdate bychromatography on deoxycorticosterone-Sepharose fol-lowed by DEAE-Sephadex chromatography. The molybdatewas then removed from the purified receptor preparations byagarose gel filtration resulting in the transformation of thecomponent 1 receptor to a nuclear binding form (28). Thiscomponent 1 contains the A subunit as described by Schraderet al. (29). Only component 1 and not component 2 receptorspecies (28) was used in these studies since it was obtainablein larger quantities and bound the nuclear acceptor sites moreextensively than component 2.

Isolation of NAP and DNA. Nuclei and chromatin wereisolated and purified using modifications of methods de-scribed (7, 9, 13, 16). All steps were performed at 4°C. Theisolation and characterization of the native NAP has beendescribed (8, 12, 13). The chromatin and NAP were resus-pended in a solution containing 4 mM Tris'HCl/0.2 mMEDTA, pH 7.5/0.5-1.0 mg of DNA/ml. They were stored at-80°C until needed.DNA was isolated from hen spleen nuclei as described (8,

13, 16). Extreme care was taken during the DNA isolation toprevent damage to the DNA or NAP, which can affect PRbinding. The final product, in NTE buffer (10 mM Tris HCl,pH 7.4/10 mM NaCl/1 mM EDTA), was analyzed for purityby measuring DNA with the diphenylamine reaction (30),RNA by the orcinol reaction (31), and protein by the method

Abbreviations: NAP, nucleoprotein complex isolated from chroma-tin; NAPf, nuclease-digested NAP; DNAf, DNA isolated from NAPf;PR, progesterone receptor.*To whom reprint requests should be addressed.tCobb, A. & Leavitt, W. W., 67th Annual Meeting of the EndocrineSociety, June 19-21, 1985, Baltimore, p. 83, no. 330 (abstr.).

8839

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Proc. Natl. Acad. Sci. USA 83 (1986)

of Lowry et al. (32) or Bradford (33). An acceptable DNApreparation contained quantities with less than 1.0% proteinor RNA with respect to DNA.

Nuclease Digestion of NAP. NAP was resuspended inDNase I digestion buffer (100 mM Tris HCl, pH 7.4/10 mMMgCl2) at a concentration of 1 mg of DNA per ml at 4TC for2 hr. The NAP was then centrifuged at 100,000 x g for 30 minin a Beckman TL-100 microultracentrifuge. For nuclease di-gestion, it was found that this "prespinning" of NAP resultedin a more consistent preparation of digested NAP. The pelletwas resuspended, and an aliquot was assayed forDNA content.The remainder was quick frozen in a dry ice/ethanol bath andlyophilized. This lyophilized material is stable when stored atroom temperature. For digestion, NAP was resuspended inDNase I buffer, at 1 mg ofDNA per ml, and digested for varioustimes using 300 units of DNase I per mg ofDNA. The digestionwas terminated by the addition ofEDTA to 20mM followed bycentrifugation in the Beckman TL-100. The pelleted NAPfragments (NAPf) were washed once, and an aliquot was takenfor DNA measurement. The remainder was lyophilized andstored at room temperature with no significant reduction in PRbinding over the course of 2 months.We have also utilized an alternative procedure for NAPf

preparation that minimizes the degradation of the DNA in theNAPf preparations with no measurable differences in PRbinding observed when compared with the DNase I proce-dure. This procedure, a modification of that used by Wu (34),was not used extensively due to the high cost of the bulkenzyme treatments. In this procedure, whole NAP wastreated with 10 units of DNase I per mg of DNA at 12TC for30 min in DNase I digestion buffer to yield a partially digestedNAP. To this was added a 0.1 vol of lOx exonuclease IIIdigestion buffer (0.66 M Tris HCl, pH 8.0/6.6 mM MgCl2/10mM 2-mercaptoethanol) and digestion occurred with 100units of exonuclease III per mg of DNA at 30°C for 10 min.The NAP was then recovered by centrifugation for 30 min at105 x g in the microultracentrifuge, washed once withS1-nuclease digestion buffer (40 mM sodium acetate, pH4.5/1 mM ZnSO4/250 mM NaCl), and then resuspended inthis buffer at half the original volume. Si-nuclease digestionwas performed with 5000 units of S1 nuclease per mg ofDNAat 37°C for 30 min. Following centrifugation, the pellet ofNAPf was washed twice with 20 mM Tris HCl, pH 7.5/1 mMEDTA and then lyophilized.For the isolation of the DNA associated with NAPf

(DNAf), the NAPf was resuspended in 50 mM Tris HCl, pH7.5/1 mM EDTA at 1 mg of DNA per ml and treated for 30min at 37°C with RNase A (100 ,g/ml), followed by a similartreatment with Pronase (100 ,ug/ml). Following phenol ex-traction, the DNAf was recovered by ethanol precipitation.PR Binding to Nucleoprotein Complexes. The streptomycin

filter binding method for assessing the binding of steroidreceptors to nuclear components has been reported by thislaboratory (9) and was modified to improve the yields ofDNAf in each assay. For binding purposes, NAPf wasresuspended at 1 mg ofDNA per ml in NTE buffer at 4°C for2 hr with gentle homogenization. The binding assays wereperformed in silanized Microfuge tubes in a total volume of1 ml. The conditions for binding were identical to thosealready published (9) except that 20 ,ug of DNA (as DNAf,NAPf, or DNAf per assay) was used. Binding was initiated bythe addition of the [3H]PR and was allowed to proceed for 45min at 4°C. The PR-DNA complexes were precipitated by theaddition of streptomycin sulfate to 0.2% followed by incu-bation for 15 min at 4°C. The precipitated complexes werecollected by centrifugation for 10 min in a BeckmanMicrofuge and washed three times with 0.02% streptomycinsulfate in NTE buffer. The [3H]P was extracted with 1 ml of95% (vol/vol) ethanol at room temperature for 1 hr. Thesamples were centrifuged, and the supernatants were added

to 10 ml of a 2:1 (vol/vol) mixture of Phase CombiningSystem (Amersham) and xylene (PCS xylene) for measure-ment of radioactivity. Background values from tubes con-taining the same amount of [3H]PR but no NAP weresubtracted from the assays containing NAP and the same[3H]PR levels. The concentration ofDNA was determined bythe diphenylamine reaction (30). Recoveries of input DNAwere consistently between 70% and 80%.

RESULTSWhen NAP or DNA was digested with DNase I, there was anincrease in the percent of acid soluble material, reaching aplateau at 15-20 min of digestion. In the case of DNA,digestion ultimately resulted in a greater than 90% conversionto acid soluble material. On the other hand, when NAP wassimilarly digested, there was conversion to only about 40%acid solubility. Thus, some protection of the DNA boundwith protein was suggested. Supporting this is the fact thatthe recovery of fragments of DNAf from NAPf (i.e., thedigested native NAP) as described was found to be 8-12% ofthe DNA from nondigested NAP after the 2-15 min ofDNasedigestion. No recovery of DNAf from digestion of the pureDNA was found. As shown in Fig. 1 A and B, when NAPf,generated by a 2-min DNase I digestion, was bound by PRand the binding was corrected for the binding due to the DNAffrom the NAPf, the PR binding per mass of DNA wasmarkedly increased. The uncorrected (total) binding to NAPfand NAP did not change. Only the amount ofDNA per assaydecreased. The level ofPR binding to NAPf was consistentlyenhanced 4- to 5-fold over PR binding to native NAP (Fig. 1B)and enhanced 20- to 25-fold greater binding than to intactchromatin on a per mass ofDNA basis. As shown in Fig. LA,the PR binding to DNAf from the NAPf, isolated after 2 min

4- ~~~~~~~~4

02- D0ase

0

0l B D

Time of DNase digestion, mmn Untreated Exo III/Si

FIG. 1. PR binding to nuclease-treated NAP. The partiallypurified avian oviduct PR was bound to nuclease-digested oviductNAP. (A) PR binding to DNase I digestion of NAPs (Su) or the DNAsfrom these NAPs (in) for the times indicated is shown uncorrected.(B) PR binding to the DNase I-digested NAP, corrected for thebinding to the respective DNA (isolated from the same NAPf), isshown. (C) PR binding to NAPs (an, some of which were digestedwith DNase, Exo III, and 51 nuclease, or to the DNAs (in), some ofwhich were isolated from these NAPs is shown. (D) Specific binding(corrected for the binding to the respective DNA bindings) is shown.In all experiments, the data are presented as the mean ± SD of thethree replicate experiments.

8840 Biochemistry: Hora et al.

Proc. Natl. Acad. Sci. USA 83 (1986) 8841

ofDNase I digestion, was only slightly increased over that tonative DNA. Thus, the PR binding to the DNAf remainsbelow that to the NAPf and does not play a role in theenhanced PR binding to NAPf. On occasion, more extensivedigestion with DNase I (i.e., 5-min digestion) resulted indamaged DNAf with the expected increase in PR binding toDNAf (35) (see Fig. lA). However, the binding to NAPfgenerated by the 5-min digestion is still greater than that to itsDNAf. Even more extensive nuclease digestion of NAPresults in a loss of PR binding (data not shown).

Fig. 1 C and D shows PR binding to NAPf and DNAfgenerated by the alternate (milder) method of limited DNaseI digestion, followed by exonuclease III and finally S1enzyme treatments. PR binding to NAPf is increased 10-foldover that of native NAP while PR binding to DNAf remainsthe same as that to native DNA (Fig. 1C). As shown in Fig.ID, the specific binding ofPR to NAPf is 20-fold higher thanthe binding to native NAP. Since this latter method ismarkedly more costly than the former (DNase I) method, theformer method was generally the method of choice. In anycase, the nucleoprotein acceptor sites appear to be resistantto nuclease action, and the digestion procedure seems toenrich these sites.

Protection of fragments ofDNA by tightly bound proteinsin NAP was further verified by agar gel electrophoresis of theDNAf from NAPf. As shown in Fig. 2, small DNA fragmentscan be isolated from NAP following digestion with DNase I.No bands were observed when pure DNA was digested andsimilarly analyzed (data not shown). The NAPf showed someRNA contamination, as seen by comparing the gel lanes withand without added RNase A (Fig. 2). Binding studies withRNase-treated NAP showed a slight reduction in binding, butthis reduction was similar for both the NAPf and DNAf,indicating a nonspecific RNA binding by the receptor (datanot shown).The saturability of the PR binding to NAPf was next

examined. It has been shown that the binding of rather crudepreparations of PR or estrogen receptor to native chromatinor NAP was saturable and of high affinity (7-11, 17, 20, 36).

I4 6 i I 9 1 ()

FIG. 2. Agarose gel electrophoresis ofthe DNA from the digestedNAPs. A typical agarose gel of the DNA from 0- (lanes 6 and 8), 2-(lane 7), and 5- (lane 9) min digestions of the NAP with DNase I. Forthe 0 time, EDTA was added at 20 mM before the addition of DNaseI. After digestion, the products were phenol extracted, ethanolprecipitated, and electrophoresed through 1.4% agarose gels in TBEbuffer (0.9 M Tris base/0.9 M boric acid/0.025 M EDTA, pH 8.3).Parallel samples [0- (lanes 2 and 4), 2- (lane 3), and 5- (lane 5) mindigestion] were also treated with RNase A at 100 Ag/ml beforeanalysis. The standards are HindIlI-digested phage X DNA (lane 1)and the 123-base-pair ladder (lane 10) from Bethesda ResearchLaboratories.

As shown in Fig. 3A, the binding of this rather cruderadioactive PR preparation to NAPf appeared to be saturablewhen titrated with increasing quantities of this preparation.The saturation was substantiated by separate experimentsshowing that this nuclear binding can be competed by theaddition of nonradioactive PR composed of unlabeled pro-gesterone bound to the similar receptor preparation (Fig. 3B).The latter binding assay included only 150 ,.d of [3H]PR perassay to permit a sufficiently high quantity of nonradioactivePR to be added to the same assay and to emphasizecompetition only at the highest affinity sites (see Resultsbelow). The binding ofPR to DNAf was the same at all molarratios of PR/[3H]PR tested, (i.e., nonsaturable) and dis-played a reduced level of binding compared to that to NAPf.

O3 Ax

z

0

0 200 400 600[3H]PR added. Wl

B°15t

0~

0 1 2 3 4 5PR/[3H]PR added, vol/vol

FIG. 3. Receptor binding to the nuclease-digested NAP bypartially purified PR. (A) PR titration of binding to NAPf is shown.The NAPf was prepared by digestion with DNase I and was analyzedfor PR binding. Various amounts of PR were added to each bindingtube containing 20 ,ug ofDNA as NAPf (0) orDNA (o). DNA bindingat similar volumes of added PR was also determined. The differencebetween NAPf binding and DNA is also shown (x). Each pointrepresents the mean ± SD of triplicate analyses, and the data arerepresentative of many experiments. (B) Competition between[3H]PR and unlabeled PR for binding to NAPf is shown. NAPf wasderived from a 15-min digest of NAP. DNAf was isolated from NAPfand also bound with [3H]PR in the presence of various amounts ofunlabeled PR. For each point, 150 ,ul of [3H]PR and various volumeratios of nonradioactive PR (similar to the activated [3H]PR butbound with nonradioactive progesterone) were bound to NAPf or toDNAf. No competition for DNAf binding was seen, and the [3H]PRbinding values were subtracted from the NAPf binding values. Allbinding experiments were performed at a constant protein concen-tration of 1 mg/ml, with bovine serum albumin added to make up anydifferences between labeled and unlabeled PR. The data are ex-pressed as the mean + SD of triplicate analyses, are corrected forDNAf binding, and are representative of a number of experiments.

Biochemistry: Hora et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

When the competition studies were performed using freenonradioactive progesterone and the NAPf, no competitionwas observed, indicating a requirement for PR complex (datanot shown). Similar binding results were observed using themilder (DNase/exonuclease/Sl nuclease) digestion method.Thus, the saturable, high-affinity binding of PR to NAPfdisplayed similar properties as did the PR binding to intactNAP (8, 9, 11) or chromatin (7, 36).To assess for any effects due to contaminants in the

receptor preparation, the binding to NAPf was measuredusing the highly purified component 1 of the oviduct PR (28).Fig. 4 shows that this purified PR also binds the NAPf in asaturable pattern. The binding of component 1 to NAPf isapproximately 10-fold greater than that to whole NAP (datanot shown). As shown in Fig. 4B, Scatchard analysesidentified two classes of binding sites similar to that reportedfor the native NAP and chromatin (8, 10, 20, 36). The highestaffinity class of sites displayed a kd of 0.2 nM and the secondclass a Kd of 2.8 nM. Using the Rosenthal correction method(37), the properties of the highest affinity class of sites cor-rected for the influence of the second class of sites indicatesaKd of0.85 pM and involves approximately 23,900 moleculesbound per cell nucleus (Fig. 4B). This value is well within thevalue calculated for PR binding to intact NAP (8, 10, 20, 36),indicating that practically all of the PR acceptor sites arepreserved during the nuclease treatments. Further supportfor a saturable binding was demonstrated by competitionstudies using unlabeled progesterone bound to the activated,purified receptor. Fig. 5 shows data from a series of exper-iments as assessed by Scatchard analyses. The data show thatthe highest affinity class of sites can readily be competed byactivated component 1 of the purified receptor bound bynonradioactive progesterone. The highest affinity class ofsites is eliminated by the addition of a low level of nonra-dioactive PR (Fig. 5B). At higher quantities of nonradioactivePR (C and D), the second class of sites is abolished. Thesedata confirm those in Figs. 3 and 4 that the PR binding to thesenuclease-resistant sites is saturable, of high affinity, andresembles those measured in nontreated chromatin and NAP(8, 10, 20, 36).

DISCUSSION

Although the exact nature of the native high-affinity nuclearacceptor sites is unknown, the data presented in this com-

8A2f

6

X 0.2

0

0.1

O.

Q 0.

0)0 .

0O

O.

0 so 160 0 80Bound [3H]PR, pM

160

FIG. 5. Binding of NAPf with purified PR (component 1) in thepresence of excess nonradioactive receptor. NAPf containing accep-tor sites for PR was incubated with increasing levels of [3H]PR-component 1 either with or without the addition of the component1-PR (unlabeled) that was prepared with a mixture of 19 nMprogesterone and 1 nM [3H]progesterone. All binding experimentswere conducted, and the resultant data are presented as described inFig. 4. Binding at control conditions (A) was determined using[3H]PR (component 1), alone with the addition of ovalbumin (0.78mg/ml) to adjust for total protein. In other studies the binding of[3H]PR (component 1) was competed by the addition of unlabeled PR(component 1) at 15 I&l (0.20 pmol) (B), 50 ,ul (0.66 pmol) (C), and 100gl (1.32 pmol) (D). Data from the competition studies were correctedfor the contribution of radioactivity from unlabeled PR (component1) that had been labeled at a low specific activity for [3H]proges-terone. All points represent the average of duplicate analyses.

munication and others support the hypothesis that the nu-

clear acceptor sites for PR in the avian oviduct are

deoxyribonucleoprotein complexes. Further, it appears thatthese binding sites are protected from DNase I digestion. Asecond (milder) method of digestion, utilizing a combinationof DNase I, exonuclease III, and Si-nuclease, followed themethod ofWu (34) to map protein binding in Drosophila heatshock genes. This latter (milder) method also yields DNAfragments of 100-200 base pairs that are protected fromnuclease activity and exhibit enriched PR binding. UsingNAPf prepared by both methods of digestion, the PR bindingis shown to be saturable, to be competed by unlabeled PR, tobe of high affinity, and to require an intact receptor. Also,

0.5 1.0 2 3 0 40 80 120[3H]PR added, pmol Bound [3H]PR, pM

160 200

FIG. 4. Titration of binding of NAPf with purified progesterone-receptor component 1. NAP was digested with DNase I for 5 min andprepared for binding. Highly purified PR (component 1) was prepared as described (28). (A) Fragments from a 5-min digest ofNAP were incubatedwith 0.01-2.33 pmol of [3HJPR-component 1. Each point represents the average ± SEM of duplicate samples. (B) *, Data from A are resolvedby Scatchard analyses into two classes of binding components with Kdl, 0.181 nM; N1, 5.63 x 104 molecules per cell; Kd2, 2.79 nM; N2, 2.98X 10 molecules per cell. o, Projection of the data by the method of Rosenthal (37) reveals a specific binding component with Kdp, 84.9 pMand Np, 2.39 x 104 molecules per cell.

A B

. 0

.0

C D

O haA-A-

8842 Biochemistry: Hora et al.

Proc. Natl. Acad. Sci. USA 83 (1986) 8843

given the fact that binding to NAPf is substantially greaterthan to either NAP or DNA alone, we conclude that theacceptor sites for the avian oviduct PR are nuclease resistantand that nuclease digestion provides a useful first step in theenrichment of the "native" (undissociated) acceptor sitesthat are specifically bound by the progesterone receptor inthis system. It is interesting to note that component 1 of thehighly purified receptor exhibits marked binding to NAPf,relative to NAP. This binding to NAPf displays a saturable,high-affinity binding that is competible with unlabeled PR.That component 1 exhibits marked binding to NAPf isconsistent with binding studies performed with component 1and whole nuclei (18). Component 1 contains the A subunitofthe oviduct PR described by Schrader et al. (29). However,the higher binding of component 1 (A subunit) to NAPf is notconsistent with other studies (35), also supported by studiesin this laboratory, that demonstrated that the A speciesprimarily binds the DNA and the B species binds thechromatin.Based on their nuclease resistance, these nuclear acceptor

sites may be related to those associated with the nuclearmatrix. Currently, the matrix is defined as a salt-, detergent-,and nuclease-resistant substructure of the nucleus. Thepresence of newly synthesized RNA (39) and DNA (38) onthe nuclear matrix leads to the speculation that this structurehas functional significance in the nucleus. With regard tosteroid hormone action, the nuclear matrix has been reportedto contain tightly and specifically bound steroid receptors(40, 41), similar to that observed with PR binding to oviductacceptor sites (10, 20, 36). The presence of these tightlybound receptors in the nuclear matrix may provide a focalpoint for the primary interaction of receptors with thenucleus. However, any relationship of the acceptor sitesdescribed in this paper and those within the nuclear matrixare only speculative at this point. First, the two nuclearentities are isolated differently. Second, the matrix DNA isdifficult to isolate free of protein; and, third, the receptors arenot dissociated from the matrix by high salt and detergents(40, 41). The latter two properties differ from the acceptorsites described in this paper.The nuclease-resistant sites described in this paper may

allow a rough assessment of their location with regard tostructural genes. Two general models can be constructed.First, the acceptor sites may be located near the structuralgenes that they regulate. In this case, the nucleoproteinacceptor sites may serve to only accumulate the receptor inthe nucleus (i.e., a nonfunctional reservoir-type site). Thiswould be followed by a shift of the receptor (or its subunit)to sites neighboring the structural gene (i.e., to a functionalsite) as suggested by other investigators (29, 42). In thesecond model, the acceptor sites themselves may be part ofa distant regulatory gene(s). In this case, the steroid-receptorcomplex may interact with this region inducing the synthesisof a regulatory RNA that may be translated into a regulatoryprotein (trans-acting agent) that binds to the importantcontrol elements in the 5'-flanking regions of distant struc-tural genes. The induced regulatory RNAs themselves maybe regulatory controlling elements and, acting as anti-senseRNAs, bind to the 5'-flanking regions of structural genes.This model is speculative and awaits purification of theacceptor sequences, the demonstration of their spatial ori-entation relative to structural genes, and evidence for veryearly sex steroid-induced regulatory RNAs or mRNAs pre-ceding the steroid-induced mRNA of standard structuralgenes.

We gratefully acknowledge Ms. Kay Rasmussen and Ms. VickieBauer for their technical assistance and Ms. Beth Allred for her

excellent clerical assistance. This work was supported by GrantsHD9140 and HD16705 from the National Institutes of Health and theMayo Foundation.

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