Ludwig Institute for Cancer Research, Inselspital, 3010 Bern, Switzerland. Communicated .... sites of the Bluescript polylinker to create pbs/3c(-2300/. +487)CAT.
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 104-108, January 1989 Biochemistry
Prolactin and glucocorticoid hormones synergistically induce expression of transfected rat 13-casein gene promoter constructs in a mammary epithelial cell line (growth hormone/insulin/inducible enhancer/terminal differentiation/milk protein gene regulation)
WOLFGANG DOPPLER*, BERND GRONER*, AND ROLAND K. BALL* Ludwig Institute for Cancer Research, Inselspital, 3010 Bern, Switzerland
Communicated by Elwood V. Jensen, October 3, 1988
ABSTRACT We have detected hormone response elements in the promoter region of the rat f3-casein gene that confer the synergistic action of prolactin and glucocorticoid hormones upon transcription of chimeric gene constructs. A 2800base-pair (bp) rat (3-casein gene fragment containing 2300 bp of 5' flanking sequence was placed in front of a chloramphenicol acetyltransferase (CAT) reporter gene and stably transfected into the mouse mammary epithelial cell line HC11. Addition of prolactin or dexamethasone alone was sufficient for a modest induction of the fusion gene. The simultaneous presence of both hormones produced a strongly synergistic effect, which did not require the presence of insulin. Induction of the fi-casein-CAT gene was only observed in stably transfected confluent cell cultures. Analysis of a 5' deletion series of the j3-casein-CAT gene construct revealed a stepwise loss of hormone inducibility; 285 bp of 5' flanking sequence was sufficient to mediate the synergistic action of lactogenic hormones on expression. The response was reduced by half when compared with the construct containing 2300 bp of the 5' flanking region. Synergistic inducibility further decreased in deletion mutants between -285 and -265 and was completely abolished between -180 and -170. Thus, the 5' flanking region between -285 and -170 contains cis-acting sequences, which are required for mediating the effect of prolactin and dexamethasone.
Recently, we described the isolation of the cloned mammary epithelial cell line HC11 (10). It was derived from the BALB/c mouse mammary epithelial cell line COMMA-1D (11) and is unique in maintaining the capability to produce the major mouse milk protein f3-casein under the control of lactogenic hormones. HC11 cells do not require cultivation on exogenous extracellular matrix or cocultivation with fibroblasts or adipocytes for efficient induction of /-casein protein synthesis. Prolactin increases the transcription rate of the endogenous P-casein gene (10). We tested the potential of HC11 as a recipient cell line for the analysis of the lactogenic hormone control of milk protein gene expression by gene transfer methods. The 5' flanking sequences of the rat ,B-casein gene were recombined with the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HC11 cells. A strong induction of CAT expression by lactogenic hormones was observed in stably transfected HC11 cells. The induction was dependent on prolactin, was enhanced by dexamethasone, and required sequences located upstream of nucleotide -170 of the ,Bcasein gene. Thus, the milk protein ,3-casein is regulated at the level of transcription and requires the synergistic action of two different classes of hormones. This study provides a basis for a molecular description of the cis- and trans-acting elements used in control of the transcription of milk protein genes by lactogenic hormones.
The lactogenic hormones prolactin, hydrocortisone, and insulin have been shown to regulate milk protein expression (1). Induction of efficient milk protein synthesis by these hormones is restricted to the terminally differentiated mammary epithelium. Complex cell-cell and cell-hormone interactions are required for terminal differentiation (1, 2). Important insights into the mechanisms by which hormones and other agents control the expression of genes have been gained by the introduction of transfected genes and promoter-gene constructs into cultured cells (3, 4). Such studies have led to the definition of a number of DNA sequences or response elements mediating the effect of these inducers. Most of these response elements, such as those for interferons, cAMP, serum growth factors, heavy metals, heat shock, or steroids (3, 5-7), reside in the 5' flanking region of the induced genes and have the properties of inducible enhancers. The strategy to introduce milk protein gene constructs into cultured mammary epithelial cells and monitor hormonal effects on their expression, to date, has been unsuccessful. Cell lines isolated from the mammary gland either lose their capability to produce milk proteins when kept in culture or require growth on extracellular matrix components (8, 9).
MATERIALS AND METHODS Construction of CAT Plasmids. A 2.8-kilobase (kb) EcoRI/EcoRI fragment of the genomic rat /8-casein gene was kindly provided by J. M. Rosen (12). It consists of 2.3 kb of 5' flanking sequence, the first noncoding exon, and 0.45 kb of the first intron. The EcoRI sites were converted into BamHI sites by filling in the 3' recessed ends with the Klenow fragment of Escherichia coli DNA polymerase I and adding BamHI linkers (13). The fragment was cloned into the BamHI site of pBLCAT3 (14) in the same orientation as CAT to create pfpc(-2300/+487)CAT (see Fig. 1). The numbers in parentheses indicate the 5' and 3' borders of 83-casein gene sequences, according to the numbering given in ref. 12. The 5' deletions were introduced in the sequencing vector pBluescript KS+ (Stratagene, LaJolla, CA). A 4.4-kb Xba I/Kpn I fragment containing the 2.8-kb rat ,8-casein fragment, CAT, and simian virus 40 sequences was excised from pI8c(-2300/+487)CAT and reinserted into the Xba I/Kpn I sites of the Bluescript polylinker to create pbs/3c(-2300/ +487)CAT. Using the exonuclease III/mung bean nuclease technique (Stratagene; ref. 15) progressive 5' deletions were created starting from the HindIll site at -332 of the rat
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: CAT, chloramphenicol acetyltransferase. *Present address: Friedrich Miescher Institut, P.O. Box 2543, 4002 Basel, Switzerland. 104
Biochemistry: Doppler et al. /3-casein. Bluescript sequences were protected by cleaving the Sac I site. Deletion end points were determined by double-strand DNA sequencing (16). Cell Culture and Hormones. Cells were grown in RPMI 1640 medium with 10% heat-inactivated fetal calf serum, 5 Ag of insulin per ml, 10 ng ofepidermal growth factor per ml, and 50 Ag of gentamycin per ml (growth medium) (10). The medium was changed every 2 days. Epidermal growth factor was always omitted during lactogenic hormone induction. Ovine prolactin, bovine insulin, and dexamethasone were obtained from Sigma; human growth hormone (iodination grade) was from UCB-Bioproducts (Braine-L'Alleud, Belgium). Transfections. Cells were transfected by the calcium phosphate precipitation technique (17). Then, 1-2 x 105 cells were plated on a 9-cm culture dish. One day later, culture medium was changed to Dulbecco's modified Eagle's medium with 10%o fetal calf serum and cells were cotransfected with 10 ,g of plasmid DNA and 1 Ag of pSV2-neo (18). Medium was changed to growth medium (see above) 1 day later and replaced the following day by the same medium containing 200 ,ug of G418 per ml. Usually 100-1000 G418-resistant colonies were obtained per 9-cm dish. The cells were pooled after treatment with trypsin and expanded to 107 cells in G418-containing growth medium. The expanded cell pool was used for the hormone induction experiments. RNase Protection Analysis. Total cytoplasmic RNA was isolated (19) and subjected to RNase protection analysis (10). SP6-transcribed probes were generated from the Pvu IIlinearized plasmids pSP65-/3c-HS and pSP65-pc/CAT-SP. These plasmids were created by inserting the 0.7-kb HindIII/Spe I and the 0.3-kb Spe I/Pvu II fragment from pI8c(-2300/+487)CAT into HindIII/Xba I or Xba I/Sma I double-digested pSP651, respectively (20). CAT Assays. Cells were harvested by treatment with trypsin, washed twice with phosphate-buffered saline, and suspended in 0.25 M Tris HCl (pH 7.8). Extracts were prepared by four cycles of freeze-thawing. The lysed cells were centrifuged at 10,000 x g for 5 min and the supernatant was heated for 10 min at 60°C. Denatured protein was pelleted at 10,000 x g for 5 min and aliquots of the supernatant were taken for protein determinations (21) and assay of CAT enzyme activity, essentially as described (22). [14C]Chloramphenicol (20 ,uM) (Amersham) was used in the incubation mixture and the reaction products were resolved by thin-layer chromatography. Conversion was determined by cutting out the radioactive spots of nonacylated and acylated forms of chloramphenicol and measuring radioactivity by liquid scintillation counting.
RESULTS Transcription of a Chimeric f-Casein-CAT Gene Is Induced by Lactogenic Hormones in HCll Cells. To examine whether DNA sequences responsible for the lactogenic hormone control of P-casein expression are located in or around the promoter region, we recombined a 2.8-kb restriction fragment from the 5' end of the rat ,B-casein gene with the bacterial CAT gene (Fig. lA; see Materials and Methods). This construct ppc(-2300/+487)CAT was cotransfected with the neomycin-resistance gene contained in pSV2-neo into the mammary epithelial cell line HC11. Two antisense RNA probes were made in vitro from fragments cloned into the pSP65 vector. The probe HS (Fig. LA) spans the first exon of the f3-casein gene. If the pf3c(-2300/+487)CAT construct is initiated and spliced as the rat 13-casein gene is (12), a fragment comprising the first exon should be protected from RNase digestion (Fig. 1A). Two signals of 43 and 46 nucleotides were found in protection experiments in which RNA from stably transfected HC11
Proc. Natl. Acad. Sci. USA 86 (1989)
105
A .
rat B-casein (-2300/+487)
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CAT
.
-..
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-309
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-180 -
r-160
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FIG. 1. Structure and hormonal control of transcripts of
pfSc(-2300/+487)CAT stably transfected into HC11. (A) Probes
used in RNase protection experiments. Initiation and splice donor sites were mapped by the SP6 probe HS. The HS probe of 916 nucleotides (nt) contains 707 nt derived from the rat /3-casein gene
(HindIII/Spe I). The probe SP contains the 298 nt of p,8c(-2300/ +487)CAT between the Hindil site in the /-casein sequence and the Pvu II site in the CAT sequence. (B) RNase protection analysis. HC11 cells were kept for 2 days in growth medium after reaching confluence and then incubated with hormones for 2 days. Total cytoplasmic RNA was isolated and 20 ,ug (probe SP, lane C) or 4 ,ug (remaining lanes) was used for RNase protection analysis. Protected fragments of probe HS were analyzed on a 10% polyacrylamide/8 M urea gel and protected fragments of probe SP were analyzed on a 6% polyacrylamide/8 M urea gel. Fragments of pBR322 digested with Hpa II and end-labeled with 32p were used as size markers (lane M). Size markers (in nt) are shown on the right. Arrows indicate the position and size of protected fragments. RNA from transfected HC11 cells treated with the following combinations of hormones was analyzed: lane N, no hormones; lane 1, insulin; lane 2, prolactin and insulin; lane 3, dexamethasone and insulin; lane 4, prolactin, dexamethasone, and insulin. In lane C, RNA from untransfected HC11 cells, induced with prolactin, dexamethasone, and insulin was analyzed. Concentrations were as follows: prolactin, 5 ,ug/ml; dexamethasone, 1 ,AM; insulin, 5 ,ug/ml.
cells treated with lactogenic hormones was used (Fig. 1B). This corresponds to the previously described exon boundaries of the ,B-casein gene (12). A second probe, SP (Fig. 1A), examined the splicing of the transcript from pf8c(-2300/ +487)CAT in induced HC11 cells. This probe spans the junction between the first intron of the ,B-casein gene and the CAT gene. A fragment of 187 nucleotides was protected, indicating the use of a cryptic splice acceptor site at position +476 of pfc(-2300/+487)CAT. The nucleotide sequence 5'-TAAAAClTTllCTAG-3' precedes position +476. This sequence is similar to the rodent splice acceptor consensus sequence (23).
106
Biochemistry: Doppler et al.
A comparison of the intensity of the signals shown in Fig. 1B indicates that the abundance of RNA transcripts from p,8c(-2300/+487)CAT is hormonally controlled. Cells treated with prolactin, dexamethasone, and insulin (Fig. 1B, lane 4) show increased levels of RNA detectable with both probes. Less p,3c(-2300/+487)CAT RNA was present in cells treated with insulin (lane 1), prolactin and insulin (lane 2), or dexamethasone and insulin (lane 3). A basal level of transcription was detectable in cells cultured in the absence of hormone (lane N). No signal was found in untransfected cells (lanes C). Synergistic Action of Lactogenic Hormones on p.ic(-2300/ +487)CAT Expression. The introduction of pf3c(-2300/ +487)CAT into HC11 cells results in accurate RNA initiation and the hormonal induction of RNA levels (Fig. 1B). The activity of the bacterial CAT gene linked to the /3-casein promoter sequence was used to quantitate the effects of prolactin, dexamethasone, and insulin on the transcriptional activity of this promoter (Table 1). Confluent cultures of ppc(-2300/+487)CAT-transfected HC11 cells were induced with various combinations of hormones for 2 days and the CAT activity was determined in protein extracts. A basal level of CAT expression was detected in cells in the absence of hormones, confirming the results in Fig. 1B. Insulin by itself had no effect on the basal activity, whereas dexamethasone or prolactin caused a 4-fold induction. Insulin enhanced the response to prolactin 2-fold but had no effect on the dexamethasone inducibility. A strong synergism was observed when prolactin and dexamethasone were added simultaneously. This effect did not require the presence of insulin. The activity was increased 37-fold above the basal activity (Table 1). Thus, signals generated by prolactin and by glucocorticoid hormones are required to cause a maximal induction of the 13-casein gene promoter. A significant induction of CAT activity was already seen 3 hr after addition of dexamethasone together with prolactin. It continued to increase over 6 days (data not shown). This is similar to the stimulation of the transcription of the endogenous /8-casein gene by prolactin (10). The CAT activity in p.Bc(-2300/+487)CAT-transfected HC11 cells was measured as a function of the concentration of prolactin, growth hormone, and dexamethasone in the medium (Fig. 2). In the presence of 10-6 M dexamethasone, a half-maximal response was obtained with 3 nM (68 ng/ml) ovine prolactin or 1 nM (22 ng/ml) human growth hormone. Both hormones have been shown to bind with similar affinities to the prolactin receptor (24). Cells kept in 220 nM (5 ,ug/ml) ovine prolactin showed a half-maximal response to Table 1. Effect of lactogenic hormones on expression of p/3c(-2300/+487)CAT transfected into HC11 CAT activity,* Hormone added Induction nmol per min per mg of protein Ins Pri Dex ratio, -fold 1 1.02 0.57 + 1.3 1.31 + 0.44 + 4.1 4.16 ± 0.55 + + 3.5 3.56 ± 0.74 + 4.1 4.12 ± 0.95 + + 8.1 8.39 ± 1.98 + + 37.5 38.21 ± 3.04 + + + 32.11 ± 3.42 31.5 HC11 cells derived from a pool of stably transfected cells were kept for 2 days in growth medium after reaching confluence and then incubated with hormones for 2 days. Concentrations are as follows: prolactin (Prl), 5 ,g/ml; dexamethasone (Dex), 1 uM; insulin (Ins), 5 ,ug/mI. *Means ± SEM of three experiments with two pools of cells derived from individual transfections.
Proc. Natl. Acad. Sci. USA 86
(1989)
*
100
T/~~~ (0~~~~~~~ 8 10 9 o 11
7
6
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6
E50E X
E 25-
0
10
11 - log
9
8
[hormone, M]
FIG. 2. Concentration requirements for ovine prolactin, human growth hormone, and dexamethasone for expression of p3c(-2300/ +487)CAT in HC11 cells. Cells pretreated as described in Fig. 1B were incubated with hormones for 2 days. Dependence of CAT expression on ovine prolactin (o) or human growth hormone (e) was determined in the presence of 1 gM dexamethasone and 5 ,ug of insulin per ml. Dependence of CAT expression on dexamethasone (n) was analyzed in the presence of 5 ,ug of ovine prolactin and 5 ,tg of insulin per ml. Bars indicate SEM of three experiments with two pools of cells from individual transfections.
dexamethasone at 8 nM, a concentration also determined for the glucocorticoid receptor-mediated effects of dexamethasone (25). The (3-Casein Gene Promoter Is Only Inducible in Confluent Cell Cultures by Prolactin and Glucocorticoid Hormones. The induction of the casein-CAT construct by lactogenic hormones was found to be dependent on the density of the cell culture as shown in Fig. 3. Treatment of exponentially
growing transfectants with prolactin, dexamethasone, and insulin for 2 days did not enhance expression of CAT protein when compared to cells treated with dexamethasone and insulin for the same time period. Hormone inducibility was only detected in cells that were confluent for at least 1 day during the 2 days of hormone treatment. The response was
166
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days after confluency FIG. 3. Dependence of the lactogenic hormone response on cell culture conditions. Confluent pools of HC11 cells transfected with pBc(-2300/+487)CAT were split 1:4 and hormones were added either immediately or after different intervals of culture in growth medium. Two days after addition of hormones, cells were harvested and CAT activity was determined. The time indicates the number of days after the cell cultures have reached confluence. e, Prolactin, dexamethasone, and insulin; o, dexamethasone and insulin. Concentrations are the same as in Fig. 1B. Bars indicate SEM of experiments from pools of cells derived from individual transfections.
Biochemistry: Doppler et al.
Proc. Natl. Acad. Sci. USA 86 (1989)
further increased in cells that had reached confluence before they received hormones. Maximal CAT expression after a 2-day stimulation with hormones was seen in cell cultures that had reached confluence 2 days before hormone addition. After cultures of HC11 cells became confluent, the culture density increased -2-fold before a maximal density was reached. Analysis of Rat (8-Casein Regulatory Sequences Required to
107
Table 2. Induction of CAT expression by prolactin and dexamethasone in HC11 cells transfected with 5' deletions of pbspc(-2300/+487)CAT Induction ratio (-fold) of CAT activity after hormone addition 5' border Prolactin and of ,-casein Prolactin Dexamethasone dexamethasone -2300 4.0 ± 0.7 22.4 ± 3.9 2.6 ± 0.3 -330 2.9 ± 0.4 1.2 ± 0.2 10.8 ± 1.9 -221 1.9 ± 0.2 0.72 ± 0.09 2.1 ± 0.4 -44 0.87 ± 0.02 0.98 ± 0.03 0.95 ± 0.09 Pools of transfected HC11 cells were precultured and incubated with hormones as described in Table 1. Induced cells received insulin plus the indicated hormones. The induction ratio of CAT activity is relative to the activity in the presence of insulin alone. Results are expressed as means ± SEM from two to four individual transfection experiments. Mean CAT activities of uninduced cells varied between 0.83 and 1.21 nmol per min per mg of protein in these experiments.
Induce CAT Expression by Prolactin and Dexamethasone. The
conferral of lactogenic hormone inducibility on the bacterial CAT gene was obtained by a 2.8-kb DNA fragment of the rat 13-casein gene. To further delimit the sequence requirements for this response, the 2.8-kb ,B-casein fragment together with CAT and simian virus 40 sequences were inserted into the sequencing vector pBluescript KS+ to create pbs/3c(-2300/ +487)CAT. The rat sequence was progressively deleted from the 5' end of this construct. Fig. 4 shows the ratios of CAT activities when constructs with decreasing lengths of 5' flanking sequence were induced with prolactin, dexamethasone and insulin, or insulin alone in transfected HC11 cells. A maximal response (22-fold) was obtained with the construct containing 2.3 kb of 5' flanking sequence. Removal of =2 kb resulted in pbslc(-330/+487)CAT, retaining 330 nucleotides 5' of the RNA initiation site. The inducibility of this construct was reduced to 10-fold. A further decrease in inducibility to -3-fold was observed in deletions retaining 265-180 nucleotides of 5' flanking sequence, and the inducibility was entirely lost in deletion mutants containing 170 nucleotides or less of 5' flanking sequence. The CAT activities of the parental construct and three of the tested 5' deletion mutants in the presence of prolactin, dexamethasone, or both hormones are shown in Table 2. The synergistic response of pbs,8c(-2300/+487)CAT was lower than the 32-fold induction in p/c(-2300/+487)CAT (Table 1), which differs only in the vector sequence. This might reflect the effect of the vector on basal transcription levels. The inducibility by prolactin alone was reduced in pbsf3c(-330/+487)CAT, while a 10.8-fold synergistic inducibility remained. No significant inducibility by dexamethasone alone was observed with this construct. pbs(3c(-221/+487)CAT showed a further decreased response to prolactin alone and only a 2.1-fold synergistic induction. pbs,8c(-44/+487)CAT was no longer inducible. These experiments indicate that multiple sequence elements are required for the lactogenic hormone response.
Sequences between -180 and -265 confer a modest response. The response is sharply enhanced by sequences between -265 and -285. Sequences between -330 and -2300 confer the inducibility by dexamethasone alone.
DISCUSSION We have shown that it is now possible to use gene transfer methods to study the lactogenic hormone control of milk protein gene expression. This was dependent on the isolation of the hormone-responsive mouse mammary epithelial cell line HC11. The 5' flanking sequences mediate the synergistic induction by dexamethasone and prolactin of the rat 8-casein promoter in HC11 cells, which have terminally differentiated under defined culture conditions. The dependence of the lactogenic hormone inducibility of HC11 cells on culture conditions is reminiscent of the more complex requirements that primary mammary epithelial cells display for maintenance of their differentiated functions (8, 9). These cells require a suitable substratum or the cocultivation with adipocytes (2) to produce milk proteins in response to lactogenic hormones. In HC11 cells, the formation of a confluent cell layer is the prerequisite to induce responsiveness to lactogenic hormones. Confluent epithelial cells stop proliferating, deposit extracellular matrix, increase cell-cell contact, and polarize. All these events might con-
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i
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iji!lt 4-
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-
-
-
-250 -200 -150 -100 -50 +1 position of 51 border of B-casein FIG. 4. Synergistic inducibility of CAT expression by prolactin and dexamethasone in a 5' deletion series of pbsl3c(-2300/+487). Pools of HC11 cells transfected with various 5' deletions of the rat 3-casein fragment (-2300/+487) in a Bluescript CAT expression vector were treated with hormone* as described in Fig. 1B. The induction ratios of CAT activity in cells treated with prolactin, dexamethasone, and insulin compared -300
to the activities in cells that received insulin only are shown by bars (see Materials and Methods). CAT activities of uninduced cells varied between 0.46 and 2.36 nmol per min per mg of protein in the individual transfected cell pools.
108
Biochemistry: Doppler et al.
tribute to the establishment of the terminally differentiated state of HC11 cells. Regulatory sequences that confer the synergistic action of prolactin and glucocorticoid hormone upon the ,3-casein gene promoter are located at least within the first 2300 nucleotides 5' of the RNA initiation site (Fig. 4). The region between -511 and +487 of the f3-casein gene promoter has recently been shown to confer tissue-specific expression in transgenic mice (J. M. Rosen, personal communication). Thus, the sequences mediating tissue-specific expression and hormone induction are contained within the same region. The stepwise reduction of inducibility shown in Fig. 4 points to the existence of discrete functional elements in this region. Various reports have documented the cooperative interaction between repeated enhancer elements or synergism between different neighboring enhancer and transcription factor binding elements (3, 26, 27). We and others have screened the functionally relevant DNA region for noteworthy features. Hall et al. (28) and Yu-Lee et al. (29) have identified conserved sequences between -160 and -110 in several milk protein gene promoters. In the rat P-casein gene, this region harbors two inverted repeats: 5'-(-145)TTTCIT-
GGGAAAGiAAAATAGAAAOAAACCATTTTCTA(-113)-
3'. The spacing of the hexameric units of these motives is 1011 nucleotides; thus, the repeated sequences are presented on the same side of the DNA helix. Such structures could be important for the coordinate action of transcription factors. The sequence between -160 and -110 was not sufficient to mediate the lactogenic hormone response (Fig. 4) but might be necessary for the function of the hormone response elements located 5' from that region. This is further supported by our observation that the f3-casein sequences between -167 and -2300 did not confer inducibility to the thymidine kinase promoter of the herpes simplex virus (W.D. and R.K.B., unpublished data). Detailed information is available on the DNA sequences that interact with the glucocorticoid receptor complex (30). A consensus receptor binding sequence TGTTCT is found at -510 in the rat 13-casein gene. As shown in Table 2, deletion of the region containing this sequence abolished the response to glucocorticoid alone. The region between -330 and +487 does not contain a TGTTCT motif, but it is sufficient to mediate the effect of glucocorticoid in the synergistic response. Here, the effect of glucocorticoids could be indirect and dependent on the synthesis or repression of proteins. We still cannot exclude a role for the small untranslated first exon of the rat /3-casein gene. However, 3' deletions up to nucleotide +46 of the 2.8-kb rat /3-casein fragment showed unimpaired hormone inducibility (data not shown), which indicates that the fragment of the first intron is not involved in mediating the effect of the hormones. In vitro mutagenesis and recombination of DNA elements, the use of specific inhibitors, and studies of DNA-protein interactions can be used to further elucidate the mechanism of lactogenic hormone regulation of milk protein gene expression. We are grateful to Dr. J. M. Rosen for critical reading of the manuscript and providing us with the rat f3-casein fragment. The authors wish to thank A. Schlafli and H. Birk for excellent technical assistance and C. Wiedmer and T. Diabat6 for typing the manuscript.
Proc. Natl. Acad. Sci. USA 86
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