Polypeptide Hormone Regulation of Gene Transcription

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2975-2979, May 1984 Biochemistry

Polypeptide hormone regulation of gene transcription: Specific 5' genomic sequences are required for epidermal growth factor and phorbol ester regulation of prolactin gene expression (pituitary/DNA-mediated gene transfer) SCOTT C. SUPOWIT*t, ELLEN POTTER*t, RONALD M. EVANSt, AND MICHAEL G. ROSENFELD* *Eukaryotic Regulatory Biology Program, University of California, San Diego, School of Medicine, La Jolla, CA 92093; and tMolecular Biology and Virology Laboratory, The Salk Institute, La Jolla, CA 92138 Communicated by Daniel Steinberg, January 23, 1984

ABSTRACT A fusion gene containing 5' rat prolactin ge- these genes by various regulatory agents outside their nor- nomic sequences ligated to the structural portion of the rat mal cellular and chromosomal environment (13, 14). By us- growth hormone gene (grl) was introduced by DNA-mediated ing transfectional analysis, it has been demonstrated that gene transfer into mammalian cells by using a chimeric plas- DNA sequences located 5' to the so-called "TATA" box and mid vector. Clonal transfected cell lines produced a mRNA RNA cap site are necessary for efficient and accurate RNA that used the authentic 5' initiation site and that was processed polymerase 11-catalyzed transcription of many eukaryotic to the predicted size. The intracellular levels of this RNA prod- genes, in vivo and in vitro (15-17). This technology also per- uct were increased 2.5- to 5-fold by exposure of the cells to mitted the evaluation of the hypothesis that specific genomic epidermal growth factor (EGF) and 2- to 3-fold by exposure of regions are responsible for hormonal and metabolic regula- the cells to a potent phorbol ester, phorbol 12-myristate 13- tion of gene expression (18-28). Transfer of regulation has acetate, apparently due to regulation at the level of gene tran- been reported in the case of steroid hormones (18-23), scription. Substitution of the 5' prolactin DNA sequences by 5' poly(rI)poly(rC) (24), and heavy metal (25) regulation of growth hormone DNA sequences resulted in the loss of EGF specific genes. inducibility. A genomic sequence in or near the 5' flanking We therefore employed DNA-mediated gene transfer to portion of the prolactin gene therefore appears to confer poly- determine whether specific genomic sequences are required peptide hormone transcriptional regulation upon the gene. for polypeptide hormone regulation upon responsive genes. A fusion gene containing 5' flanking prolactin genomic infor- Polypeptide hormones and neuropeptides represent a di- mation and growth hormone coding regions (grl) was ex- verse class of intercellular regulatory substances. They are pressed with correct initiation and RNA processing events, critical for development and homeostatic control, and they in a human cell line rich in EGF receptors. The gene product act to regulate the expression of specific genes (e.g., see was induced by the peptide hormone, EGF, apparently ex- refs. 1-6). To understand the events by which the binding of erting transcriptional effects, and by a phorbol ester. Substi- a peptide hormone to the plasma membrane receptor initi- tution of 5' prolactin genomic sequences with comparable 5' ates events that can rapidly (within 1-2 min) modulate gene sequences from a nonresponsive gene (growth hormone) transcription (4-6), it is necessary to define whether a spe- abolishes the transfer of hormonal regulation. cific genomic sequence is required and sufficient for conferring the effects of polypeptide regulators on gene transcrip- METHODS tion. Cell Culture and DNA Transfection. Human A431 cells In clonal rat pituitary tumor cell lines (GH) (7) thyrotro- were maintained in Dulbecco's modified Eagle's medium pin-releasing hormone (TRH), a hypothalamic tripeptide, containing 1% fetal calf serum. Supercoiled plasmid DNA and epidermal growth factor (EGF), a small polypeptide was introduced into cultured cells by using the calcium phos- originally isolated from the submaxillary glands of male phate precipitation technique (29) followed by a glycerol mice, increase the production of prolactin (8, 9) and prolac- shock after 4 hr (30); neomycin-resistant cells were selected tin mRNA (2-5). We have demonstrated that both EGF and by growth in the presence ofthe antibiotic G418 at 400 ,ug/ml TRH act at the nuclear level to rapidly increase the tran- (31). Individual foci of transformed cells were maintained in scription of the prolactin gene (5, 6). In addition, phorbol the presence of G418 at 200 ,Ag/ml. esters produce effects on GH cells, such as cell shape Analysis of RNA and DNA. Purified genomic DNA restric- change, growth alterations, and increased prolactin biosyn- tion fragments were resolved by electrophoresis of 0.8% thesis, similar to those produced by EGF and TRH (10). We agarose gels and then transferred to nitrocellulose by the find that phorbol esters increase prolactin mRNA levels, method of Southern (32). Total cellular RNA was isolated (2) prolactin gene transcription, and phosphorylation of a basic and DNA was removed by two precipitations with 2 M nuclear protein (BRP) in a fashion comparable to the effects LiCl/10 mM sodium acetate, pH 5.5 (33). DNA content of all of EGF or TRH (5-11). Phorbol esters such as phorbol 12- RNA samples was determined by a fluorometric assay using myristate 13-acetate (PMA) appear to directly activate a the DNA dye Hoechst 33258 (34). Samples (10,g) were ana- Ca2'- and phospholipid-dependent protein kinase, referred lyzed by fractionation using denaturing gel electrophoresis, to as protein kinase C' (12). blotting, and DNA-excess hybridization as described (4). DNA-mediated transfer of regulated eukaryotic genes into The 5' end of the growth hormone cDNA-reactive RNAs heterologous mammalian cells provides the requisite analyt- were analyzed by using an appropriate DNA probe radiola- ic technology for studying the expression and induction of beled by replacement synthesis with T4 DNA polymerase

The publication costs of this article were defrayed in part by page charge Abbreviations: TRH, thyrotropin-releasing hormone; EGF, epider- payment. This article must therefore be hereby marked "advertisement" mal growth factor; PMA, phorbol 12-myristate 13-acetate; SV40, in accordance with 18 U.S.C. §1734 solely to indicate this fact. simian virus 40; kb, kilobase(s); bp, base pair(s). 2975 Downloaded by guest on September 26, 2021 2976 Biochemistry: Supowit et aL Proc. NatL Acad Sci. USA 81 (1984) (35) and the S1 nuclease method described by Berk and Organization of the Fusion Gene DNA in Transformed Sharp (36). After hybridization with excess labeled DNA Cells. Human A431 cells were transfected with chimeric probe, the RNADNA hybrids were digested with S1 nucle- plasmids containing either the rat prolactin-growth hormone ase and subjected to electrophoresis on denaturing 8% acryl- fusion gene (pSV2ne0grl) or rat growth hormone gene amide/8 M urea sequencing gels (37). The nuclear run-off (pSV2ne0GH). Hirt DNA extracts (41) prepared from several transcription assay was performed as previously described of the stable pSV2neogrl-transfected cell lines revealed no ex- (4, 5) using 7 x 107 cpm per hybridization, and growth hor- trachromosomal neo or grl DNA. Transfected DNA se- mone cDNA was inserted into M13 phage DNA as probe. quences derived from the chimeric gene were detected in the chromosomal DNA of the transformed cell lines by analysis RESULTS of HindIII-digested genomic DNA. DNA from the parental Construction of Chimeric Rat Prolactin Genomic Clones for A431 cell line shows two strongly hybridizing high molecular Transfectional Analysis. A simian virus 40 (SV40) hybrid plas- weight (>20 kb) HindIII genomic fragments, reflecting en- mid vector pSV2neo that contained the dominant selectable dogenous growth hormone genes (Fig. 2). A unique 5.7-kb marker Tn5 phosphotransferase (neo) (31) was modified to HindIII fragment present in the intact plasmid, which spans contain a fusion gene in which the 5' regions ofthe growth hor- the entire grl gene, including 5' and 3' flanking sequences, is mone gene, a gene that is not induced by EGF, were replaced present in 6 of the 10 pSVneogrl-containing clonal cell lines with 5' prolactin genomic sequences (38, 39) in such a way analyzed, with an estimated chromosomally integrated copy that mRNA transcribed from this gene should contain the number of 1-4 in different cell lines (Fig. 2). In one cell line, information encoded in the first prolactin genomic exon and instead of the 5.7-kb band, a slightly smaller (5.1-kb) band the second through fifth rat growth hormone genomic exons was visualized (grl d). In addition, the cell lines contained a (see Fig. 1). Analysis of the sequences of the first prolactin wide variety of additional reactive HindIlI fragments ranging "donor" splice site and the first growth hormone "acceptor" in size from 3.8 to >20 kb (see Fig. 2). These heterogeneous site showed that they contained the requisite G-T ... A-G fragments are likely to have resulted from integration of the sequences under "Chambon" rules of splicing (40). There- chimeric plasmid DNA at sites within the 5.7-kb HindIII re- fore, if the prolactin gene contains a regulatory element for gion into variable integration sites of flanking A431 cellular EGF induction within or near the 5' nonflanking sequences, DNA sequences. These data indicate that most lines have the fusion gene (gri) should be productive of an EGF- or multiple integrated copies of both intact and modified grl fu- PMA-inducible mRNA containing the cap site and 82 nucleo- sion genes, integrated into a wide variety of genomic sites. tides of prolactin mRNA (exon I) followed by growth hor- One line (grl i) appears to have a single integrated copy ofthe mone mRNA sequences transcribed from exons 2 through 5 gene. of the growth hormone gene. This chimeric genomic con- RNA Analysis in Transfected Cell Lines. Eight of 10 G418- struction, designated grl, was inserted into the unique resistant cell lines analyzed produced the RNA of the size BamHI site of pSV2neo such that the two discrete transcrip- predicted for the product of the fusion gene expression (1 kb) tion units, one containing neo (the gene conferring G418 and (see Fig. 3). RNA prepared from nontransformed A431 cells neomycin resistance), under SV40 early promoter control contained no detectable hybridization to growth hormone and adjacent to an enhancer element, and the second, the grl cDNA probes. All of the cell lines that contained at least one fusion gene, are in opposite transcriptional orientations. A copy of the intact grl gene produced the authentic 1-kb RNA second plasmid, constructed by replacing the Xba I fragment product of the fusion grl gene (Fig. 3). The several lines that prolactin DNA in the grl gene with the original Xba I frag- contained only rearranged grl genomic integrants produced ment of growth hormone, reconstituted the intact rat growth RNAs of inappropriate size, with one exception (grl d). An hormone gene (21). S1 nuclease resistance assay (36) was used to prove that the RNA product in transfected cell lines was initiated at the 5' Prolactin Growth hormone 3' A431 a b c d e f 9 h i B X '-_ X B kb

B AmpI@C 2 3 2SV2neogr 2.3---_ 1

pBR322 ori pSV40 ori

FIG. 1. Structure of the transducing vectors. The vectors are de- 57_ rivatives of pSV2no (generous gift of Peter Southern); pBR322 is represented by the solid black segment of the circle; the hatched segment specifies the 1.4-kilobase (kb) neo gene, which is preceded 4.3-'- by the SV40 origin of replication (ori) and early promoter (small stippled region); the SV40 small tumor antigen intervening sequence and an SV40 polyadenylylation sequence are schematically represented by the large stippled region. AmpR, ampicillin resistance. In- serts were either the 7.8-kb BamHI fragment containing the rat 2.2-- growth hormone gene (33) or the diagrammed fusion gene. In the fusion gene, rectangles indicate exons and the lines indicate introns. FIG. 2. Chromosomal DNA (20 ,ug) isolated from parental A431 A 3.8-kb Xba I fragment of the prolactin gene containing exon I [82 cells and clonal cell lines transduced with the grl plasmid was digest- base pairs (bp)], 750 bp of intron A, and 3.0 kb of 5' flanking se- ed with HindIII and the restriction fragments were separated on quences (38, 39) was ligated into the Xba I sites of the rat growth 0.8% agarose gels. After transfer to nitrocellulose the blotted DNA hormone gene. Restriction endonuclease sites: B, BamHI; X, Xba I; was hybridized to 32P-labeled growth hormone cDNA. Size markers R, EcoRI; and P, Pst I. were HindIlI-digested X phage DNA. Downloaded by guest on September 26, 2021 Biochemistry: Supowit et aL Proc. NatL. Acad. Sci. USA 81 (1984) 2977

correct site, using a 111-bp Pst I fragment of the prolactin cultures, with a maximal induction (3.5-fold) at 16-24 hr. gene, which includes the entire 82 bp of exon I and contains There is subsequently a progressive decrease in grl RNA 5' and 3' flanking sequences, such that renatured DNA levels; however, even at 48 hr grl RNA remains above the would be larger than DNA protected by formation of levels observed in untreated cells (Fig. 3B). A comparison of RNADNA hybrids (see Fig. 4). An excess of the DNA these data with the EGF induction of authentic prolactin probe was hybridized to the RNA samples from control and mRNA in GH4 cells shows that the kinetics of prolactin in- EGF-induced cells, and the predicted 82-bp fragment that duction in response to EGF are very similar in both cell corresponds to exon I of the authentic rat prolactin gene was types, with a tv2 for maximal induction (4-fold) of approxi- protected (Fig. 4), suggesting that the appropriate initiation mately 7-9 hr in the pSV2n,,grl-transfected cell lines, com- site was used in the transcription of the grl gene. No protec- pared to 5-8 hr in GH cells (Fig. 5). These data suggest that tion was observed when RNA samples from nontransformed the kinetics of EGF transcriptional effects and the stabilities A431 cells were substituted in the hybridization reaction. of the mature mRNA transcripts are quite similar for the en- EGF Stimulates Increase of grI mRNA Levels in Stably dogenous gene in rat pituitary cells and the transfected fu- Transfected Cell Lines. As shown in Fig. 3A the levels of sion gene in a human cell line. S1 nuclease mapping con- RNA reactive with growth hormone cDNA from several grl- firmed that EGF induced the accumulation of an mRNA spe- transformed cell lines (a-e) were consistently increased by cies that used the correct 5' initiation site, protecting the 2.5- to 5-fold by treatment of the cells with EGF. The kinet- identical 82-nucleotide fragment as authentic prolactin ics of grI mRNA accumulation in response to hormone in- mRNA (Fig. 4). S1 nuclease resistance analysis revealed that duction are shown for one of the cell lines (pSV2neogrl i, Fig. the levels of grl mRNA in clonal pSV2neogrl-transfected cells 3A). An increase in the growth hormone cDNA-reactive (pSV2neogrl i) were approximately 1/40ih of the levels of au- RNA can be detected at 6 hr after addition of EGF to cell thentic prolactin mRNA in GH4 cells (prolactin mRNA is approximately 1% of the total mRNA in this cell line). Be- cause the neo transcription unit and its adjacent SV40 en- A hancer element were physically linked to the grl gene in the a b c d e x y plasmid used for transfectional analyses, the potential co-

M C - + bp f loo

': '.' 1.0-_* *o 06. kb I

1 10- -

90- _

B c 8276 Of_ 0 8 18 30 48 0 8 18 30 48 67- _

1 5-_ kb 10 34- CAP pstl Pstl - RLCI 5'tYFPROLACTIN EXN 3'

FIG. 3. Blot analysis of RNAs from transformed cell lines. (A) 1 1 1bp Total cellular RNAs were extracted from clonal A431 cell line trans- fectants containing either the pSV2ne0grl (a-e) or pSV2neOGH (x and FIG. 4. S1 nuclease resistance analysis of the grl fusion gene y) DNAj and aliquots (10 gg) were electrophoresed on denaturing RNA and authentic prolactin mRNA, using a 5'-specific DNA 1.5% agarose/formaldehyde gels and transferred to nitrocellulose. probe. A 32P-labeled 111-bp Pst I fragment of the rat prolactin gene The hybridization probe was a 32P-labeled growth hormone cDNA. (5 x 104 cpm) was hybridized to 20 ,g of total RNA from GH4 cells The individual cell lines were either mock treated (-) or treated (+) (C) or to 50 ,ug of total RNA from control (-) or EGF-treated (20 hr) with 10 nM EGF for 20 hr. (B and C) Total cellular RNA from a (+) clonal cell lines (pSV2n,0grl i). The fragment contains the entire pSV2n,,grl-transformed cell line (pSV2nr~grl i) was extracted from first exon of the prolactin gene (82 bp) flanked by 5' and 3' chromo- plates either mock treated (0) or treated for various times (8, 18, 30, somal sequences (see schematic at bottom). RNA-DNA hybrids or 48 hr) with EGF and analyzed as described above, using 32p_ were digested with S1 nuclease and electrophoresed under denatur- labeled growth hormone cDNA (B) or a 32P-labeled Pvu II fragment ing conditions. Lane M contains Msp I-digested labeled pBR322 as of the neo gene (C). markers. Downloaded by guest on September 26, 2021 2978 Biochemistry: Supowit et aL Proc. NatL Acad Sci. USA 81 (1984)

A 400 -

300 -

C C.)0

c g 200- 0 60--4-,looi z 0 E z 100- E

Control EGF PMA PMA 200k 24 hr 24 hr 48 hr

FIG. 6. Effect of phorbol esters on expression of the grl gene in clonal transfected cell lines. The relative levels of grl mRNA (open bars) and neo mRNA (hatched bars) were quantitated by densito- 1 00l metric scanning of the autoradiograph after RNA blot analysis of 10- 12 18 24 30 ,ug RNA aliquots prepared from control and EGF- (10 nM, 24 hr) or PMA- (100 nM, 24 and 48 hr) treated i cell cultures. Time, hr pSV2,egrl imately 2.5-fold 24 hr after addition to the cell cultures (Fig. FIG. 5. Kinetics of mRNA accumulation in response to treat- 6). EGF treatment for 24 hr in the identical cell line resulted ment of cell cultures with EGF. (A) Accumulation of grl RNA in the clonal transformed cell line pSV2negrl i after treatment with EGF in a 4.0-fold increase of the fusion gene mRNA. Further- (10 nM) for various times. (B) Prolactin mRNA accumulation in more, the levels of this RNA species remained at approxi- EGF-treated (10 nM) GH4 cells. mately the same levels at the 48 hr time point, in contrast to the attenuation observed after treatment with EGF (Fig. 3B). The effects of PMA are gene specific, because the levels of regulation of the neo gene by EGF was evaluated. The levels neo mRNA in identical RNA samples in which the fusion of the 1.5-kb neo mRNA remained unaltered, or even slight- gene was induced were unchanged between control and EGF ly decreased, in this and other lines during the time course or PMA-treated cells (Fig. 6). of EGF treatment (Fig. 3C). Thus, whatever the effects Transcription of the grl Gene in Transfected Cell Lines. If on it of SV40 enhancer grl gene expression, does not gene transcription were the critical determinant of grl gene confer EGF responsiveness. These results indicate that the expression, then the fact that grl RNA is 1/30th to 1/40th of pSV2neogrl-transformed cell lines synthesized an EGF-in- prolactin mRNA in GH cells would lead one to predict that ducible mRNA that is reactive with growth hormone cD)NA the rate of transcription is approximately 1/40th. In GH cells of the predicted size, while synthesis of neo mRNA was untreated with EGF, the prolactin gene transcription rate is ap- changed. Further evidence that the 5' portion of the prolac- proximately 15-20 ppm/kb. Similar analysis was done with tin gene contained the determinants critical for EGF regula- clonal line pSV2neogrl i of the A431 transfected cell lines. tion was obtained by the observation that no EGF regulation The transcription rate of the grl gene was quantitated by us- was observed in clonal transformant cell lines containing a ing 7 x 107 cpm of labeled run-off transcripts. Hybridization growth hormone gene in which the 5' prolactin DNA se- was below the level of accurate detection (<0.1 ppm/kb) for quences of the fusion gene were replaced by those from the unstimulated cultures, and increased to 0.3-0.4 ± 0.1 rat growth hormone gene (see Fig. 3A, lanes x and y). ppm/kb for EGF-treated (50 min) cells in two independent Copy number of the transfected DNA does not appear to experiments, a transcription rate consistent with the relative be the critical determinant of the level of expression of grl contents of grl and prolactin RNAs in A431 and GH cells, mRNA, because clone if which appears to contain a single respectively. These analyses support the prediction that copy of transfected DNA, produces the highest level of grI EGF rapidly increases grl gene transcription in a fashion RNA. The a fact that EGF induces growth hormone cDNA- analogous to the effects of this hormone on prolactin gene reactive RNA in every cell line, despite the apparent vari- expression in rat pituitary (GH) cells (4). ability of chromosomal insertion sites and copy number, sug- gests that sequences in the 5' end of the prolactin gene, rath- er than surrounding chromosomal sequences or structure, DISCUSSION confer EGF regulation upon the integrated, transfected Defining the mechanisms by which regulatory substances gene. modify gene expression and therefore alter the phenotype of Regulation of grl Gene Expression by Phorbol Esters. We the cell is fundamental for achieving an understanding of de- have noted in GH cells that the phorbol ester PMA mimics velopmental and physiological homeostasis in higher eukary- the effects of TRH and EGF on prolactin gene transcription otes. In the case of regulatory agents that bind to receptors and that protein kinase C' activation is associated with hor- within the cell, such as steroid hormones, it was predicted, monal induction of prolactin gene expression (11). There- by analogy with events previously described in the control of fore, the effects of a phorbol ester (PMA) on grI mRNA lev- expression of bacterial operons, that the receptor-ligand els were investigated in one of the transfected cell lines complex would bind to specific genomic sites and modify (pSV2n,.grl i). PMA increased the levels of grI RNA approx- gene transcription. Because of the availability of cloned ste- Downloaded by guest on September 26, 2021 13iochemistry: Supowit et aL Proc. NatL. Acad. Sci. USA 81 (1984) 2979 roid hormone-regulated genes and the introduction of DNA- 6. Granner, D., Andreone, T., Kazuyuki, S. & Beale, E. (1983) mediated gene transfer technology, support for this hypothe- Nature (London) 305, 549-551. sis has been obtained. Thus, steroid hormone regulation of 7. Martin, T. F. J. & Tashjian, A. H., Jr. (1977) in Biochemical could be transferred to fusion genes Actions ofHormones, ed. Litwack, J. (Academic, New York), gene expression by the pp. 270-317. 5' noncoding portion of the regulated gene (e.g., see refs. 17, 8. Schonbrunn, A., Krasnoff, M., Westendorf, J. M. & Tashjian, 26, and 27). A. H., Jr. (1980) J. Cell Biol. 85, 786-797. In the case of polypeptide hormones, which bind to plas- 9. Johnson, L. K., Baxter, J. D., Vladavsky, I. & Gospo- ma membrane receptors, there has been no conceptual darowicz, D. (1980) Proc. Natl. Acad. Sci. USA 77, 394-398. framework by which to explain their rapid (within 1-2 min) 10. Osborne, R. & Tashjian, A. J., Jr. (1981) Endocrinology 108, effects on specific gene transcription (4-6). Because it is es- 1164-1170. tablished that modification of specific nuclear proteins wide- 11. Murdoch, G. H., Evans, R. M. & Rosenfeld, M. G. (1984) in ly distributed in chromatin occurs rapidly in response to Biochemical Actions ofHormones, ed. Litwack, J. (Academic, polypeptide hormones (e.g., see refs. 5 and 42), models that New York), in press. 12. Nishizuka, Y. (1983) Trends Biochem. Sci. 8, 13-16. predicted either general alterations in chromatin structures 13. Benoist, C. & Chambon, P. (1981) Nature (London) 290, 304- or binding of modified proteins to specific genomic se- 310. quences were consistent with available data. The data pre- 14. Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Urlaub, G. sented in this manuscript, based upon transfectional analysis & Chasin, L. (1979) Proc. Natl. Acad. Sci. USA 76, 1373- of fusion genes, are consistent with the hypothesis that spe- 1376. cific 5' prolactin genomic sequences are responsible for con- 15. Shenk, T. (1981) Curr. Top. Microbiol. Immunol. 93, 25-40. ferring EGF regulation upon the prolactin gene and provide 16. McKnight, S. L. & Kingsbury, R. (1982) Science 217, 316-324. the initial evidence for the action of specific 5' genomic regu- 17. Breathnach, R. & Chambon, P. (1981) Annu. Rev. Biochem. latory sequences in mediation of polypeptide hormone ac- 50, 349-359. 18. Lee, F., Mulligan, R., Berg, P. & Ringold, G. (1981) Nature tions on gene transcription. It should be noted that the A431 (London) 294, 228-232. cells used in these analyses are actually growth-inhibited by 19. Buetti, E. & Diggelmann, H. (1981) Cell 23, 335-345. addition of EGF to cultures (43), an effect that EGF also 20. Kurtz, D. T. (1981) Nature (London) 291, 629-631. exerts in GH cells, where it also stimulates prolactin gene 21. Doehmer, J., Barinaga, M., Vale, W., Rosenfeld, M. G., transcription. This effect on growth is in contrast to the EGF Verma, I. & Evans, R. M. (1982) Proc. Natl. Acad. Sci. USA receptor contents of the two cell lines, which are 2 x 106 and 79, 2268-2272. approximately 1-2 x i05 receptors per cell, respectively. In 22. Robins, D. M., Park, I., Seeburg, R. & Axel, R. (1982) Cell 29, addition, the prolactin gene is transcriptionally regulated by 623-631. phorbol esters, appear to activate a Ca2+- and phos- 23. Page, M. J. & Parker, M. G. (1983) Cell 32, 495-502. which 24. Canni, D. & Berg, P. (1982) Proc. Natl. Acad. Sci. USA 79, pholipid-dependent protein kinase, referred to as C' kinase 5166-5170. (12). Because the 5' end of the prolactin gene also transfers 25. Mayo, K. E., Warren, R. & Palmiter, R. D. (1982) Cell 29, 99- regulation by phorbol esters, it is tempting to speculate that 108. both specific genomic sequences and protein modification 26. Dean, D. C., Knoll, B. J., Riser, M. E. & O'Malley, B. W. are requisite for PMA regulation of specific gene transcrip- (1983) Nature (London) 305, 551-554. tion. 27. Huang, A. L., Ostrowski, M. C., Bernard, D. & Hager, G. L. These data indicate that a human cell line possesses all of (1982) Cell 27, 245-255. the requisite biochemical machinery to transcriptionally reg- 28. Davidson, E. H., Jacobs, H. T. & Britten, R. J. (1983) Nature ulate a rat gene 5' se- (London) 301, 468-470. hormonally responsive (prolactin 29. Graham, F. L. & Van der Eb, A. J. (1973) Virology 52, 456- quences) and to accurately process the RNA transcripts, 467. suggesting a high degree of conservation between species of 30. Parker, B. A. & Stark, G. R. (1979) 1. Virol. 31, 360-369. the genomic sequences and regulatory molecules that are re- 31. Southern, P. J. & Berg, P. (1982) J. Mol. Appl. Gen. 1, 327- sponsible for polypeptide hormone control of gene expres- 341. sion. 32. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. 33. Beach, L. R. & Palmiter, R. D. (1981) Proc. Natl. Acad. Sci. USA 78, 2110-2114. 34. P. T. & R. The experiments reported in this manuscript were supported by Labarca, Paiger, U. 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