Proc. Natl. Acad. Sci. USAVol. 74, No. 5, pp. 1816-1820, May 1977Biochemistry

Regulation of growth hormone messenger RNA by thyroid andglucocorticoid hormones

(gene regulation/cell-free translation/RNA'cDNA hybridization)

JOSEPH A. MARTIAL, JOHN D. BAXTER, HOWARD M. GOODMAN, AND PETER H. SEEBURGMetabolic Research Unit, and the Departments of Medicine and Biochemistry and Biophysics, University of California, San Francisco, California 94143

Communicated by I. S. Edelman, January 21, 1977

ABSTRACT Thyroid and glucocorticoid hormones stimulategrowth hormone synthesis in cultured rat pituitary cells (GC).We have compared changes in growth hormone production andmRNA in these cells. Triiodothyronine (10 nM) and dexameth-asone (1 MtM) stimulated increases in growth hormone produc-tion by 2.5- and 3.8-fold, respectively. There were correspondingincreases in the capacity of RNA from hormone-treated cells todirect synthesis of pregrowth hormone in a wheat germ cell-freetranslation system, suggesting hormone-regulated increases ingrowth hormone mRNA. Hormone-induced changes in mRNAwere also demonstrated by determining the kinetics of hy-bridization of a cDNA probe prepared from RNA enriched(about 70%) for growth hormone translational activity with RNAfrom control and hormone-treated cells. These results suggestthat thyroid and glucocorticoid hormones can regulate growthhormone production by influencing the levels of its mRNA.

Thyroid and glucocorticoid hormones profoundly influencedevelopment, differentiation, and metabolism (1-3). Althoughlittle is known about the mechanism of thyroid hormone action,the discovery of nuclear receptors (4, 5) that are DNA-bindingproteins (6) and Tata's report (7) of changes in total RNA syn-thesis in response to thyroid hormone have directed attentionto chromatin as one possible site of hormonal control. Further,recent cell-free translational data have shown that hepatic a2uglobulin mRNA is increased in thyroid hormone-treated ani-mals (8, 9). This system is somewhat complicated because atleast four hormones are required, the kinetics are slow, and thestimulations have been performed in animals in which primaryhormone influences have not been differentiated from secon-dary influences. Nevertheless, these data may indicate thatthyroid hormones regulate specific mRNAs. In a few cases, theidea that glucocorticoids influence specific mRNAs has beensupported directly by experiments in which cell-free translationassays of mRNA, such as that for tryptophan oxygenase (10),were used, but cDNA-RNA hybridization assays have not yetbeen performed except in the case of viral RNA production (11,12).Thyroid and glucocorticoid hormones stimulate growth

hormone production in cultured rat pituitary cells (refs. 13-16;S. S. Papavasiliou, J. A. Martial, K. L. Latham, and J. D. Baxter,unpublished data). The thyroid hormone effect is blocked byinhibiting RNA synthesis, but an intermediate necessary forstimulation of growth hormone synthesis can apparently ac-cumulate in response to thyroid hormone while protein syn-thesis is blocked (16). These findings, although indirect, suggestthat thyroid hormone may regulate growth hormone mRNA.Bancroft, Wu, and Zubay (17) demonstrated cell-free synthesis

of growth hormone using mRNA from these cells in a Krebs IIascites system. By using the wheat germ cell-free system, theseworkers synthesized "pregrowth hormone" that was precipi-tated by antibodies to growth hormone and was cleaved bytrypsin into peptides that migrated on paper electrophoresiswith those from growth hormone (18). In the present studies,we have used a subline of these cells (GC cells) (17, 18) to si-multaneously study hormonal regulation of growth hormonesynthesis and mRNA.

MATERIALS AND METHODSCell Culture and Hormone Stimulation. GC cells (kindly

provided by Dr. Carter Bancroft) were grown at 370 in mo-nolayer culture in minimal essential-Joklik medium (CellCulture Facility, University of California, San Francisco),supplemented with 15% horse serum, 2.5% fetal calf serum, and0.4 mM CaC12 at 5% CO2. For hormonal stimulation, the me-dium was replaced by "hypothyroid" medium (minimal es-sential-Joklik medium containing 0.4 mM CaC12 and 10%serum from a thyroidectomized calf, obtained from RocklandFarms, Inc., Gilbertsville, PA). After 48-72 hr, the medium wasreplaced by fresh hypothyroid medium containing 10 nM tri-iodothyronine (T3) (Sigma), 1 ,uM dexamethasone (Sigma), orno hormone (control). Standard incubations were 35 hr for T3and 96 hr for dexamethasone. Ten hours before harvesting, themedium was replaced by fresh hypothyroid medium containingthe hormones at the same concentrations. GH4 cells (13) (kindlyprovided by Dr. Armen Tashjian, Jr.) were grown as monolayersin Dulbecco's modified Eagle's medium (Cell Culture Facility,University of California, San Francisco) supplemented with15% horse serum and 2.5% fetal calf serum.Growth Hormone Assay. Growth hormone production was

measured by determining [by radioimmunoassay (19)] theamount of growth hormone excreted into the medium duringthe 10-hr period before the cells were harvested, except for thekinetic study, where the period was 2 hr.RNA Preparation. Cells were harvested by trypsin treatment

and washed in ice-cold 25 mM potassium phosphate/0.1 MNaCl at pH 7.4. After 10 min at 00 in 10 mM NaCl/10 mMTris-HCI, pH 8.5/3 mM MgCI2 (5 ml/g of cells), cells weredisrupted by 10 strokes in a tight-fitting Dounce homogenizer.Nuclei were pelleted (800 X g, 5 min) and the supernatant so-lution was made 0.5% in sodium dodecyl-sulfate (NaDodSO4),1 mM in EDTA, and 100mM in NaCl. RNA was extracted bythe phenol/chloroform technique (20) and was precipitatedby 2.5 volumes of ethanol. The RNA pellet (10,000 X g, 30 min)was solubilized in H20 containing 0.5% NaDodSO4/50 mMNaCl/1 mM EDTA, and incubated at 370 .for 30 min with 100,qg/ml of autodigested Pronase (Sigma). After the phenol/chloroform extraction and ethanol precipitation were repeated,

1816

Abbreviations: GC and GH4 cells, subclones of rat pituitary cells inculture; T3, triiodothyronine; NaDodSO4, sodium dodecyl sulfate; SAC,formaldehyde-treated bacteria from the Cowan I strain of Staphylo-coccus aureus; Rot, RNA concentration (mole of nucleotide/liter) Xtime (s).

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the RNA pellet was solubilized in H20 (at 2 mg/ml) and storedat -60°. Generally, 106 cells yielded 10 Mg of cytoplasmic RNA[measured by absorbance at 260 nm (41.6 Mg/A unit)].

Protein Synthesis In Vitro. The cell-free protein-synthe-sizing extract was prepared from raw wheat germ (Old StoneMill, Niblack Foods, Rochester, NY) (21). Reaction mixtures(40 Ml) contained wheat germ extract (16 Ml), 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) pH7.6, 2 mM dithiothreitol, 3 mM Mg acetate, 80 mM KCI, 0.1mM spermidine, 1 mM ATP, 0.02 mM GTP, 8 mM creatinephosphate, 40 Mg of creatine phosphokinase per ml, 10 MCi of[35S]methionine (400 Ci/mmol, Amersham-Searle), 20 MM eachof the other 19 amino acids, and 5 Ag of cytoplasmic RNA. After1 hr at 280, the reaction mixtures were incubated with pan-

creatic RNase (10 Mg/mi, Worthington) for 15 min at 370; totalincorporation of [a5S]methionine into protein was determinedby hot trichloroacetic acid precipitation on 3 MM (Whatman)paper (22).

Immunoprecipitation of Pregrowth Hormone. Pregrowthhormone made in the cell-free system was quantitated by im-munoprecipitation using rhesus monkey antiserum to ratgrowth hormone. This was kindly provided by Dr. TetsuoHayashida, who has described the characteristics of this anti-serum in detail elsewhere (23, 24). Antigen-antibody complexeswere collected by adsorption to formaldehyde-treated bacteriafrom the Cowan I strain of Staphylococcus aureus (SAC) pre-

pared and stored as described by Kessler (25). Nonidet P-40(0.05%, Particle Data Lab) and methionine(1 mM, Sigma) were

present during the procedures. Polysomes were removed fromcell-free reaction mixtures (100,000 X g, 90 min) and the su-

pernatant solutions containing the released polypeptide chains(about 25% of the total acid-precipitable radioactivity) were

incubated for 5 min at room temperature with 30 ,l of the SACsuspension. After SAC removal by centrifugation (8000 X g,

30 s), one part of the supernatant received 20 Ml of antiserumto growth hormone diluted 1:50 in NET buffer (150 mMNaCl/5 mM EDTA/0.02% NaN3/20 mM Tris, pH 7.4); a sec-

ond part received 20 Ml of rabbit antiserum to bovine serum

albumin (Hyland Lab, Los Angeles) diluted in the same man-

ner. After overnight incubation at 40, the samples were incu-bated with 15 Ml of SAC for 5 min at 00. SAC antibody-antigencomplexes were pelleted (8000 X g, 30 s), washed three timeswith 0.5 ml of NET buffer, and transferred to new tubes. SACcomplexes were disrupted in 50 Ml of NaDodSO4-sample buffer[2.5% (wt/vol) NaDodSO4/10% (wt/vol) glycerol/5% (vol/vol)2-mercaptoethanol/65.2 mM Tris-HCl, pH 6.8] and SAC was

removed by centrifugation (8000 X g, 30 s). Radioactivityimmunoprecipitated was measured after trichloroacetic acidprecipitation of the supernatant on 3 MM paper (22), and was

expressed as the percentage of the total radioactivity incorpo-rated into released polypeptide chains measured after the firstincubation with SAC in the absence of antibody. The per-

centage of pregrowth hormone immunoprecipitated was cal-culated by subtractir.g the value obtained with bovine serumalbumin antibody (<0.1%) from the value with growth hor-mone antibody.The SAC procedure was also used to precipitate growth

hormone and prolactin from the medium of GH4 and GCcells.NaDodSO4/Polyacrylamide Gel Electrophoresis. -5S-

Labeled proteins were electrophoresed on 12.5% acrylamideslab gels (12 cm length) (26) at 20 mA per gel for 4 hr. Gels werefixed in 100 ml of 50% trichloroacetic acid for 15 min, washedthree times (20 mineach time) in 500 ml of 7% acetic acid, anddried on Whatman 3 MM paper. Dried gels were exposed to

x-ray films (Kodak, no screen, NS2T). Radioautographs werescanned on a Beckman Spectrophotometer (Acta CIII).

Hybridization Experiments. RNA from GC cells was en-riched in growth hormone mRNA sequences by using as a signalthe cell-free synthesis of pregrowth hormone. Cells were in-duced by dexamethasone (1 AtM, 4 days) and membrane-boundRNA was isolated as described by Bancroft et al. (17). RNAcontaining poly(A) was obtained by oligo(dT)-cellulose chro-matography (27). GC cells (1010; about 20 g wet weight) yielded20 mg of membrane RNA, of which 200 ,ug contained poly(A).The latter was centrifuged (130,000 X g, 22 hr) on a 5-20%sucrose gradient in 0.1 M LiCl/10 mM Tris, pH 7.5/1 mMEDTA/0.5% NaDodSO4. RNA fractions sedimenting between10 and 13 S (compared with the sedimentation of 18 and 28SRNA from GC cells) were the most enriched in growth hormonetranslational activity: 70% of their cell-free product was pre-growth hormone, as assayed by SAC immunoprecipitation.The fractions enriched in growth hormone translational ac-

tivity as well as nonfractionated cytoplasmic RNA containingpoly(A) from the dexamethasone-induced cells were used asa template for cDNA synthesis performed as detailed elsewhere(26) with [3H]dCTP (21 Ci/mmol, Amersham-Searle) as labeledsubstrate. The cDNAs were isolated as described (28) exceptthat Sephadex G-50 instead of G-150 was used. Usually, 1 ,ugof RNA yielded 0.1 ,tg of cDNA with a specific activity of 107cpm/Mg.On polyacrylamide gel electrophoresis (29) these cDNA

preparations appeared to have a mean size of 700 to 1200 nu-cleotides as compared to restriction fragments of M13 phageDNA produced by endonuclease HaeIII (29). Self-annealingof the cDNAs was about 5%.The cDNA obtained from RNA enriched in growth hormone

translational activity was further enriched for frequent se-quences by low Rot [RNA concentration (moles of nucleotide/liter) X time(s)] hybridization with polyadenylylated RNA fromGC membrane fractions. The mixture (0.1 ,Ig of cDNA and 4,g of RNA in 100 ,l of solution containing 0.3 M NaCl/20 mMTris-HCl, pH 7/1 mM EDTA/0.02% NaDodSO4) was boiledfor 2 min and reannealed at 680 for 5 min. At this time, 15% ofthe cDNA was in hybrid (S1 nuclease resistant). After additionof 50 ,l of buffer containing 0.3 M NaCl/0. 1 M sodium acetate,pH 4.5/0.01 M ZnCl2/Sl nuclease, the mixture was incubatedfor 1 hr at 420. Reactions were terminated by adding EDTAto 2 mM. The RNA was hydrolyzed with 0.6 M NaOH and thecDNA was isolated as described above.

Hybridization reactions were carried out as described else-where (30). The zero time control values were subtracted fromeach value, and the results are expressed as percent of controlwithout SI nuclease. Rot values were calculated by assuminga Rot of 12 mol-s/liter for 1 mg/ml of RNA incubated for 1 hrand were corrected to standard conditions (31).

RESULTSInduction of Growth Hormone Production in GC Cells.

GC cells respond to physiological concentrations of thyroidhormones (Papavasiliou et al., unpublished data); maximalresponses were observed at 5-10 nM T3. Fig. 1 shows thatstimulation by T3 is maximal by 30 hr and that GC cells alsorespond to the glucocorticoid dexamethasone at a concentration(1 uM) that is maximally effective in most systems (3) (maximaleffect after 60 hr). In both cases, growth hormone productionincreases within a few hours. Growth hormone production canbe directly related to synthesis of the peptide since storage ofgrowth hormone by the cells and degradation of the hormonein the cells or medium are negligible (33). Further, Samuels and

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Dexamethasone0

$ 300

0Ez 200

00

10020 40 60 80

Time (hours)FIG. 1. Kinetics ofT3 and dexamethasone stimulation of growth

hormone production. GC cells were grown in petri dishes (20 cm2).Media were replaced 2 hr before the time points. At the times indi-cated, samples were taken and frozen at -60°. Growth hormone wasnot affected by the freezing step. It was measured within 10 days andnormalized for the amount of protein (32) in the cells scraped fromeach plate. At the beginning of the experiment, there were about 105cells per cm2; these were producing about 100 ,ug of growth hor-mone/24 hr per mg of protein. Each point represents the mean ofduplicate plates.

Shapiro (16) demonstrated directly that the effect of T3 in GH,cells is due to stimulation of growth hormone synthesis.

RNA-Directed Cell-Free Synthesis. Under our conditions,upon addition of cytoplasmic RNA from GC cells, the wheatgerm system shows a maximal 25-fold stimulation of amino-acidincorporation into protein that is linear for at least 1 hr. Theincorporation is also linear with respect to the RNA added (fromhormone-treated or control cells) up to 0.2 mg of RNA perml.

Immunoprecipitation of Growth Hormone and PregrowthHormone. Specificity of the antibody used is illustrated usingGH4 rather than GC cells because the former produce bothprolactin and growth hormone. Radioactively labeled proteinsreleased by GH4 cells into the medium were immunoprecipi-tated with different antibodies. The immunoprecipitates wereanalyzed by electrophoresis on NaDodSO4/polyacrylamide gels(Fig. 2). The growth hormone antibody precipitate containsonly one identifiable protein that comigrates with iodinatedrat growth hormone. Antiserum to prolactin only precipitatedprolactin and antiserum to bovine serum albumin failed toprecipitate either growth hormone or prolactin. As expected,medium from GC cells also showed only one protein band thatwas precipitated by antiserum to growth hormone.When products synthesized in the cell-free system in response

to GC cell RNA were incubated with antiserum to growthhormone, only one protein was precipitated (Fig. 2); no de-tectable precipitation occurred in control experiments usingantiserum to bovine serum albumin. The electrophoretic mo-bility of the material precipitated by antiserum to growthhormone was that expected for the "pregrowth hormone"(molecular weight 24,000) described by Sussman et al. (18),using bacteriophage T4 proteins as molecular weight markers(34).

Quantitative Immunoprecipitation of Pregrowth Hor-mone Synthesized In Vitro. With constant amounts of growthhormone antibody, the radioactivity immunoprecipitated isrelated linearly to the added amount of product synthesized ina cell-free system. Linearity with respect to the amount ofcell-free products synthesized in response to GC cell RNA wasalso obtained when these products were mixed in different

pGH- --*__ -PL-_ ,__ _-GH _

1 2 3 4 5 6 7 8 9

FIG. 2. Radioautographs of NaDodSO4/polyacrylamide gels afterelectrophoresis of proteins excreted into the media and synthesizedin the cell-free system. (1) '25I-Labeled growth hormone. (2-6) Afterincubation of GH4 cells for 2 hr in methionine-free medium withadded [35S]methionine (1 ,uCi/ml), labeled proteins released into themedium were examined: (2) precipitate with antiserum to prolactin;(3) no treatment; (4) precipitate with antisera to prolactin and growthhormone; (5) supernatant medium after precipitation with antiserumto growth hormone; and (6) precipitate with antiserum to growthhormone. (7-9) The released chains obtained after translation ofRNAfrom GC cells: (7) precipitate with antiserum to bovine serum; (8) notreatment; or (9) precipitate with antiserum to growth hormone. GH,growth hormone; pGH, pregrowth hormone; PL, prolactin.

proportions with those synthesized in response to dog pancreasRNA under conditions in which the total amount of radioac-tivity prior to precipitation was made constant. In the samerange in which the total protein synthesis is linear, immu-noprecipitable pregrowth hormone was also linearly relatedto the amount of RNA added to the cell-free system. These dataindicate that cell-free translation coupled with SAC immu-noprecipitation can be used to measure the relative translationalactivity of growth hormone mRNA.Hormonal Effect of Growth Hormone mRNA Activity as

Measured by Cell-Free Translation. The cell-free translationproducts from cytoplasmic RNA from control and hormone-treated cells were analyzed by immunoprecipitation. There wasa good correlation between the hormonal influence on growthhormone production* and the capacity of the RNA to directcell-free synthesis of pregrowth hormone (Table 1). Thus, theactivity of growth hormone mRNA is stimulated by both classesof hormones.

As seen in Fig. 3, NaDodSO4/polyacrylamide gel electro-phoresis of the cell-free products indicates that in addition topregrowth hormone, RNA from hormone-treated cells directsan increase in the synthesis of at least three other high-molec-ular-weight polypeptides (migrations approximately 1.8, 2.2,and 3.2 cm; Fig. 3); however, synthesis of the majority of thedetectable polypeptides is not noticeably affected.Hormonal Effect on Levels of Polyadenylylated RNA

Measured by Hybridization. The cDNA probe prepared fromRNA enriched in growth hormone translational activity washybridized to an excess of cytoplasmic RNA isolated fromhormone-treated and untreated cells: it reacts faster with RNAfrom treated cells than with RNA from untreated cells (Fig. 4).From profiles such as those shown in Fig. 4, the Rot1/2 valuesobtained (50% hybridization) were used to calculate the foldincrease of the induced RNA species. The changes (Table 1) aresimilar to those of cellular and cell-free growth hormone syn-thesis.

* The lots of serum from thyroidectomized calf used in our studiescontained variable amounts of thyroid hormones. Thus, the stimu-lations observed may represent only an increase over that alreadyelicited by the endogenous thyroid hormones.

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Table 1. Influence in GC cells of thyroid andglucocorticoid hormones on growth hormone production,growth hormone mRNA activity, and RNA complementary

to a cDNA probe prepared from RNA fractionsenriched in growth hormone translational activity*

Growth hormoneHybridi-

Exp. Pro- mRNA zationTreatment no. duction activity to cDNA

None 1-5 1.0t 1.0t 1.0§T3 1 3.5 2.1 2.7

2 2.6 2.25 2.03 2.4 2.25 2.14 1.35 1.25 ND¶

Mean 2.5 2.0 2.3

Dexameth-asone 1 5.5 4.3 3.8

4 2.5 3.2 2.85 3.5 2.0 ND

Mean 3.8 3.2 3.3

* Cells were treated with T3 or dexamethasone. Growth hormoneproduction, mRNA activity, and the kinetics of hybridization ofRNA to the cDNA probe were assayed as described in the text.Values are expressed relative to controls.

t Growth hormone production in the control cells ranged from 4.3 to17.2 pg/106 cells per 24 hr.[[35S]Methionine incorporated into immunoprecipitable product inthe cell-free system by using RNA from control cells averaged 0.80%of the total radioactivity in the released chains.

§ The Rotj12 for hybridization of the cDNA probe prepared from RNAenriched in growth hormone translational activity (Materials andMethods) with RNA from control and hormone-treated cells wasdetermined. The Rotl/2 for control cells ranged from 9.2 to 13.1 mol-s/liter in various experiments. The fold stimulation is the ratio ofthe Rotl/2 of the hormone-treated samples to the control Rotj/2 (seeFig. 4).ND, not determined.

Fig. 4 also shows the hybridization of the probe made fromtotal cytoplasmic polyadenylylated RNA isolated from dexa-methasone-treated cells to excess RNA from hormone-treatedand untreated cells. The reactions are indistinguishable in theRot range examined. The hybridization occurs at higher Rotvalues and has a broader range than those with the cDNAprepared from RNA enriched in growth hormone translationalactivity, indicating that the latter probe had been enriched forcertain RNA species.

DISCUSSIONIn the present studies, cell-free translation in wheat germ ex-tract, coupled with an immunoprecipitation technique usingstaphylococci (SAC) was used to measure the translational ac-tivity of growth hormone mRNA. The SAC procedure that weused in these studies appears to be a simple method, generallyapplicable to quantify immunoprecipitable products made incell-free systems. Using this assay, we found that growth hor-mone mRNA activity in GC cells was stimulated by thyroid andglucocorticoid hormones. The stimulations were paralleled bysimilar increases in growth hormone production.From our results, some estimation of the complexity of the

hormonal effects on mRNA can also be made. When the cell-free products of the RNA from treated and untreated cells wereanalyzed by NaDodSO4/polyacrylamide gel electrophoresis,the general patterns were similar; only a few polypeptides werenoticeably affected. These data suggest that the hormonal in-

U

0 j ~~~~Dexamethasone0 5 0

CentimetersFIG. 3. Spectrophotometric scans of radioautographs of the

NaDodSO4/polyacrylamide gel electrophoresis of the products ob-tained after cell-free translation ofRNA from control and hormone-treated cells. The upper panel shows the position of the pregrowthhormone precipitated by the growth hormone antibody.

fluences are directed at a subset of the expressed genes in thissystem. There may, of course, be hormonal effects that are notdetected by the method used, e.g., in cases where mRNA levelsand consequently the proteins coded by these RNAs are too lowto be detected.To further study hormonal effects on RNA, we used hy-

bridization techniques. Two types of cDNA were synthesized.The first cDNA was complementary to nonfractionated poly-adenylylated RNA from dexamethasone-treated cells. Over therange of Rot examined, this probe hybridized with the samekinetics to cytoplasmic RNA from hormone-treated or controlcells. Thus, the hormones do not increase all polyadenylatedRNA species; any influences must be on a subset of them. Bycontrast, the second cDNA, complementary to polyadenyly-lated RNA enriched (about 70%) for growth hormone transla-tional activity, showed a striking difference in its kinetics of

0-

N-o-D._

Ir

10I l0'

Rot (mole x sec/liter)

FIG. 4. Kinetics of hybridization of total cytoplasmic RNA fromcontrol and treated cells with two different cDNA probes: cDNA madefrom cytoplasmic polyadenylylated RNA (0, control; *, T:,; *,dexamethasone) and cDNA prepared from RNA enriched in growthhormone translational activity as described above (0, control; A, T:j;o, dexamethasone). S1 nuclease background (5-8%) has not beensubtracted.

00

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80Dexamethasone',,

60 /,* T3ontrol

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1820 Biochemistry: Martial et al.

hybridization with RNA from hormone-treated or control cells.These data demonstrate that thyroid and glucocorticoid hor-mones increase the copy number of certain polyadenylylatedRNA (presumably mRNA) species.The hybridization data presented do not demonstrate that

growth hormone mRNA sequences are induced by the hor-mones. However, this may be the case since the probe wasprepared from RNA fractions in which 70% of the translationalactivity was for pregrowth hormone and the increase in copyreflected by the probe (calculated by the change in Rot1/2) issimilar to the change in growth hormone translational activity.Further, the fact that the hybridization is complete over 2logarithm units of Rot (Fig. 4) suggests that this second probewas either nearly homogeneous for growth hormone or thatsome other polyadenylylated RNA species also induced by bothclasses of hormones were present in concentrations similar tothat of growth hormone mRNA.

Because thyroid hormone receptors and glucocorticoid-receptor complexes are found in the chromatin (3, 35, 36), itis possible that the changes in mRNA levels are due to hormonalregulation of transcription, as appears to be the case with sexsteroids (37, 38). However, it is not known, particularly forthyroid hormones, whether control of mRNA is generally re-sponsible for other actions of the hormones. Nevertheless, thecurrent studies support the idea that one way thyroid and glu-cocorticoid hormones act is by controlling the levels of specificmRNAs; they also indicate that growth hormone productioncan be controlled by modulation of growth-hormone-specificmRNA.

We thank Drs. Robert Ivarie and Axel Ulrich for help. Reversetranscriptase from avian myeloblastosis virus was supplied by NCI.These studies were supported by Grants 1-RO1-AM-18878 andCA14026 from NIH, and V3/5-MB560E from FNRS (Belgium), andpreviously by Grants BC-175 from the American Cancer Society andGB-41582 from NSF. J.M. is a recipient of an ACS California Divisionfellowship, no. J-308. J.B. is an Investigator of the Howard HughesMedical Institute. P.S. is a recipient of a Deutsche Forschungs-Gem-einschaft fellowship, no. S3 306.The costs of publication of this article were defrayed in part by the

payment of page charges from funds made available to support theresearch which is the subject of the article. This article must thereforebe hereby marked "advertisement" in accordance with 18 U. S. C.§1734 solely to indicate this fact.

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