Cloning of the human oestrogen receptor cDNA

J. steroid Biochem. Vol. 24, No. Printedin Great Britain

CLONING STEPHEN

OF THE HUMAN

GREEN*,PHILIPPE WALTER*,

ELW~~D

0022-4731/86 $3.00 + 0.00 Pergamon Press L.td

1, pp. 71-83, 1986

OESTROGEN

RECEPTOR

GEOFFREY GREENEP, ANDRBE KRUST*,

JENSEN& GEOFFREY SCRACE$, MIKE WATERFIEL~

and

cDNA

COLETTE GOFFIN*,

PIERRE CHAMBON*T

*Laboratoire de G~n~tique MoI&zulaire des Eucaryotes du CNRS, Unit& 184 de Biologie Mol~culaire et de Genie GCnttique de I’INSERM, Facultk de Midecine, 67085 Strasbourg, France, TBen May Laboratory for Cancer Research, The University of Chicago, 5841 Maryland Ave, Chicago, IL 60637, U.S.A., ILudwig Institute for Cancer Research, Stadelhoferstrasse 22, CH8001 Ziirich, Switzerland and §Protein Chemistry Laboratory, Imperial Cancer Research Fund, Lincoln’s Inn Fields, London WCZA 3PX. U.K. Summary-Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lgtl0 and lgtl 1 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (J.gtl I) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (1gtlO). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, 10R3 (1.3 kbase) and IOR (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection, Furthermore, IOR contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using IOR as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER -cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.

INTRODUCTION

ERs are believed to play an important role in the growth and development of a subset of hormonedependent human breast cancers. Approximately one-third of all breast cancer tumours contain significant amounts of ERs and about two-thirds of these are able to respond objectively to some form of anti-oestrogen endocrine therapy [2,7]. Therefore a better understanding of either the mechanism of oestrogen action in these tumours, or of the way in which their expression is regulated, may lead to an improvement in the therapy of these cancers. We describe here the isolation of several cDNA clones corresponding to the mRNA sequence of the human ER.

Oestrogens

regulate gene expression in target cells through their interaction with specific receptors (for a review see Ref. [I]). The presence of oestrogen

receptors (ER) can be determined either by their high-affinity binding for tritiated oestradiol [Z] or by using specific monoclonal antibodies [3,4]. Recent studies have suggested that the oestrogen-free receptor is localized predominantly in the nuclear compartment [5,6], where it is loosely bound until its association with estradiol converts the receptor to an active form with the ability to bind tightly in the genome [2]. The activated complex is believed to act directly at some, as yet, ill-defined chromatin site(s) site(s) resulting in specific changes in gene expression, although the molecular mechanism by which ER complexes are able to modify the expression of specific genes is at present unknown. Further understanding of this mechanism has been severely hampered due to the low level of ER expression. High-level expression of ER cDNA, in both homologous as well as heteroiogous systems, should allow further insight into ER structure and function

EXPERIMENTAL

Synthesis of ds cDNA and cloning into Igt 10 and Igt I I Sucrose gradient fractions 21-23 (see Fig. 1) which are enriched in ER mRNA were pooled and used for cDNA synthesis and cloning into Agt vectors essentially as described by Huynh et al@] and Young and Davis[9, lo]. Briefly, 5 fig of enriched RNA was reverse transcribed in the presence of 35 pmol of a random primer consisting of a 13 mer synthetic oligonucleotide synthesized using a mixture of each nucleotide at each position [ 1I]. After boiling for 90 s, the second strand was synthesized with DNA polymerase I and the cDNA treated with Sl nuclease at 25°C. Any internal EcoRI sites were protected by treatment with EcoRI methylase and the extremities

at the molecular level. Since expression of the ER gene is both tissue-specific and developmentally regu-

lated, isolation of the ER gene should lead to the identification of the responsible sequence elements. “To whom reprint requests and all correspondence should be addressed. Abbreoiations: ER, oestrogen receptor; ds cDNA, doublestranded complementary DNA. 71

STEPHEN GREEN et (11.

78

of the cDNA made flush using T4 DNA polymerase. An equal mass of BcoRI linker was ligated to the cDNA overnight at 16°C using T4 DNA figase (generally 1 pig of cDNA was ligated with 1 fig of the linker GGAATTCC). The cDNA was digested with EcoRI, separated from excess linker by chromatography on a Biogel A50m column and ligated at a molar ratio of 2: 1 (cDNA:/I phage DNA) overnight at 4’C to either EcoRI-digested lgtl0 or to the EcoRT-digested and phosphatased expression vector, Igtll [8,9]. After packaging the DNA in vitro [IZ] the phage were amplified on either E. coli C600hfl (igtl0) or Y1088 (Igtll). This technique yielded approx. 5 x lo6 recombinants per pg of RNA for AgtlO and 1 x IO6 recombinants per pg of RNA for Igtl 1. Between 85 and 95% of the lgtl 1 plaques contained inserts.

1?181920212223pA* -92.5 6546-

The Lgtll cDNA library was plated onto E. coii Y1090[10], with approx. 25,000 phage per 8Scm plate. These were screened with the receptor monoclonal antibodies H222, H226, D75, D547 (1 pg/ml of each) essentially as described by Young and Davis[lO] except that proteins expressed by the plaques were transferred in duplicate onto nitrocellulose filters (soaked in IOmM isopropyi-/3D-thiogalactopyranoside) for 2 h each. Only those phage plaques producing duplicate signals were further studied. RESULTS

Sucrose gradient enrichment of MCF-7 ER mRNA Poly A+ RNA from the human breast cancer cell line, MCF-7 [13], was fractionated on sucrose gradients containing methylmercury hydroxide, and RNA sedimenting between approx. 23s and 32s was translated in vitro (Fig. 1). The proteins from each translation reaction were immunoprecipitated using a mixture of monoclonal antibodies prepared against the MCF-7 ER [3,4] and revealed by polyacrylamide gel electrophoresis and fluorography (Fig. 1). Two proteins of approx. 65 and 46 kdalton were observed. The peak of the corresponding mRNA was found in fractions 21 and 22 representing an mRNA which sedimented faster than 28s rRNA. Purified ER labelled with “‘1 co-migrated with the 65 kdalton in vitro translation component under these electrophoresis conditions (not shown) supporting the conclusion that fractions 20-23 were enriched in ER mRNA when compared with total poiy A+ RNA (lane PA+). The abundance of ER mRNA in total cellular poly A+ RNA was approx. 0.003%, as estimated by comparing the amount of [‘SS]methionine present in the immunoprecipitated protein with the amount of methionine incorporated into total protein during in vitro translation. Fractionation of poly A+ RNA on sucrose gradients

-25.7

Fig. 1. Sucrose gradient fractionation and ipl oitro translation of MCF-7 cell poly A+ RNA. RNA was isolated from the human breast cancer cell line MCF-7 using the LiCI-urea technique [14] and poly A+ RNA purified by chromatography using oligo-dT cellulose. 100,~g of poly A+ RNA was fractionated on 5-20% sucrose gradients (Il.2 ml) containing 50 mM Tris-HCl pH 7.5, 1 mM EDTA and 5 mM methylmercury hydroxide in an SW41 rotor at 40,000 rpm for 6 h and 28 fractions were collected. Aliquots from fractions K-23 were translated in t&ro in the presence of [35S]methionine (I 000 Ciimmol) using a rabbit reticulocyte lysate following the manufacturers conditions (NEN). Samples containing approx. 2 x 10b acid insoluble counts were precipitated [ 15, 161 with a mixture of the four monoclonal antibodies (H222, H226, D75, D547, 5 pg of each [3.4]). The antibody-selected proteins were then separated on a 12.5% SDS-polyacrylamide gel. Lane pA+ represents in aim translated and immunoprecipitated proteins using total poly A+ MCF-7 RNA (4 pg RNA, 16 x 10” acid insoluble counts). The sizes of the markers (BRL, high molecular weight) are shown at the right-hand side of the figure (Fhosphorylase B, 92.5 kdalton; bovine serum albumin, 68 kdalton; ovaibumin, 43 kdalton; a-chymotrypsinogen. 25.7 kdaiton).

resulted in a lo- to 15-fold purification of the receptor mRNA as determined by the same assay. Isolation of ER cDNA 0Ii~o~ucieoti~e screeprirzg

clones

by

antibody

and

Randomly primed cDNA was prepared from selected sucrose gradient fractions enriched in ER mRNA and inserted into the llgtl0 vector and the expression vector lgtl 1 (see Materials and Methods). Each of the four ER monoclonal antibodies, H222, H226, D75 and D547 [3,4] were tested separately for their ability to react with purified ER spotted onto nitrocellulose under the same conditions as those

Cloning of the human ER cDNA

used when screening the lgtI1 library. All of the monoclonal antibodies were found to react with the receptor. However antibodies H226 and IX.5 were each capable of detecting 100 pg of purified receptor, whereas in the case of H222 and D547 approx. 5 times more receptor was required to obtain the same signal (not shown). A mixture of all four antibodies was used to screen approx. l-l.5 million phage plaques. Twenty-one positive plaques were obtained of which 10 remained positive after two additional rounds of screening. On further analysis it appeared that these 10 clones represented S individual clones which were named LORO to iOR4. Two clones, 10R2 and 10R3, contained two inserted cDNA fragments, but in each case only one of the fragments corresponded to ER sequence. Figure 2 shows the reaction of each of the four monoclonal antibodies with each of the five igtl I clones. It is interesting to note that the antigen expressed by all five clones reacted only with the most “efficient” monocionai antibodies (H226 and D75). Synthetic oIigonucleotide probes were used as an alternative approach to the cloning of the ER by expression. Homogeneous preparations of the MCF7 ER were cleaved with cyanogen bromide and some of the peptides sequenced (G. Scrace, G. Greene and M. Waterfie~d: In preparation). Oligonucleotide probes corresponding to these peptides were used to screen 300,000 recombinants out of which five independent clones were isolated (AOR to 10RlO) (details to be presented elsewhere). Most importantly, one of the clones, carrying the largest cDNA insert (AORS), hybridized to the synthetic ohgonucleotides generated from two independent peptides suggesting strongly that this clone contains a cDNA insert corresponding to that of ER mRNA.

Raetfatr r

Fig. 3. Northern analysis of sucrose gradient fractions of MCF-7 poly A+ RNA. The 28 fractions from a sucrose gradient (100 pg poly A+ RNA) were pooled as indicated at the top of each lane and analysed on a 1% agarose gel containing 10 mM methyImercu~ hydroxide [17]. The RNA was transferred to diazobenzylo~ymethyl (D&M) paper and hybridized to the nick-translated cDNA insert of IORS fl81. The position of the ER mRNA is indicated at the right-hand side of the figure. The peak of 28s RNA was found in fractions 20 and 21. DNA fragments were used as size markers to estimate the length of the poly A+ mRNA.

The nick-translated insert of /20R8 was hybridized to sucrose gradient fractions of MCF-7 poiy A+ RNA (Fig. 3). A single band of approx. 6.2 kbase was seen, the peak of which appeared in fractions 23 and 24, compared with fractions 21 and 22 as found by in vitro translation of MCF-7 poly A+ mRNA from an equivalent gradient. 10R8 was used also as a probe for cross-hybridization to each of the i.OR8 clones. As shown in Fig. 4, LOR8 cross-hybridized to all of them, irrespective of the technique used for their selection, indicating that they a11contain cDNA inserts corresponding to the ER mRNA sequence.

receptor m~lonel

antibody

a@*)

“0.5 w

Fig. 2. Expression of ER antigens in the Igt I t clones. Each of the five lgtlt clones were spotted onto a plate of E. co/i Y 1090 and grown at 42°C for 4 h. The plate was overlayed with a nitrocellulose filter soaked in 1OmM isopropylfl-D-thiogalactopyranoside and left for a further 2 h at 37°C. The filter was cut into strips and each incubated with one of the four ER monoclonal antibodies using the standard assay as described for the screening (see Materials and Methods).

-03

Fig. 4. Cross-hybridization of the 10R clones with the insert c.f 20R8. Each of the isolated LOR clones was digested with E?coRl and electrophoresed on a I % agarose gel. After transfer to nitrocellulose, the filter was hybridized with the nick-translated insert of 10R8 using standard techniques [12]. Clones lOR0 to 10R4 were isolated from the lgtl 1 cDNA library using monoclonal antibodies whilst IOR5, 6, 8, 9 and 10 were isolated from a lgtl0 library using oligonucleotide probes (see text). The size of the insert of each clone is indicated at each side of the figure.

80

&EPHENGREENet al

Fig. 5. In rirro translation of hybrid-selected MCF-7 ER mRNA. The inserts from 10R3 (i.3 kbase) and lOR8 (2.1 kbase) were subdoned into pBR322 at the EcoRI sites and used 10 hybrid select ER mRNAtt9.20] using sucrose gradient fractions oFMCF-7 poiy A* RNA enriched in ER mRNA (fractions 20--23, Fig. 1). The selected RNA was translated in rirro in the presence of f%]methionine (1000 Ci/mmol) and the proteins resolved on a 12.5% SDS-polyacrylamide gel before and after immunoprecipitation using the faur ER monoclonal antibodies. Lane M represents the markers (see legend to Fig. 1).

The Iargest cfone isatated by antibody screening, (I .3 kbase), and the largest clone obtained by ohganucleotide screening, 1 OR8 (2.1 kbase), were used to select ER mRNA from sucrose gradient fractions. The mRNA selected by hybridization with either 1OR3 or 10R8 was then translated in a rabbit reticulocyte lysate system and the products electrophoresed on an ~D~-~lya~~~arnjde get before and after immunopre~ipitation with a mixture of the four monoclonai antibodies (Fig. 5). Even without immunoprecipitation a strong band of approx. 65 kdaltan was apparent in both cases, and after immunoprecipitatian only two bands of 65 and 46 kdalton were observed. These two bands carrespand to those present when total poly A+ mRNA or sucrose gradient enriched mRNA fractions were transfated in t&-o and immuno~r~~pitated (Fig. 1). These results further support the conclusion that the AOR cIanes contain ER cDNA inserts. 10R3

The human ER cDNA chicken sequences

clone cross-hydridizes with

The same amount of total poly A+ RNA isolated from faying hen oviduct, H&a, and the human

Fig. 6. (A) Northern an&d. Poly A’ RNA (30 pg) [from laying hen oviduct, WeLa cells, MCF-7 cells and T47D cells was electrophoresed on 1% agarose gels containing 10 mM methyl mercury hydroxide as described [ 171.The RNA was transferred to diazabenzyloxymethyl (D&M) paper and hybridized with the nick-translated insert ofIOR8 [18]. The arrows indicating 7.5 and 6.2 kbase represent the sizes of bands seen in the chicken and human RNA samples, respectively, using DNA fragment as size markers. [B) Sautkcm anrtfysk Genomic DNA frio fig)* digested to completion with EcoRI, was ~lecfrophoresed on a 1.5%agarose gel and transferred to dia~oben~yioxymethyl (DBMl paper [18]. The filter was hybridized with the nick-translated cDNA insert of iCH78. The sizes of the chicken and human EcoRI fragments are given on the left and right of the figure, respectively.

breast cancer cell lines MCF-7 and T47DfZI], was efectrophoresed on an agarose gel and transferred to d~azo~nzy~oxymethy? paper. When the nicktranslated IOR insert was used as a probe a singfe band corresponding to an RNA of apprax. 6.2 kbase was observed for bath the T47D and MCF-7 cell lines (Fig. 6A). The T47D cell line contains less ER than the MCF-7 ceII line when determined using a hormone binding assay PI]. The northern bIot suggests that this is also true at the mRNA level (Fig. 6A). No hybridization was observed with the human ER - ceil line HeLa. Interestingly, the huma’n ER probe was capable of hybridizing to a chicken oviduct paly A+ RNA of approx. 7.5 kbase under highstringency hybridization conditions, Further evidence of an homology between the human BR cDNA sequence and the chicken gene was obtained from Southern analysis of &OR&digested genomic DNA hybridized with the nick-translated cDNA insert of LOR8 (Fig. 6s). Such an homoIogy is also suggested from immunochemical studies [4]. Indeed, two of the four monoclonal used here (H222, H226) have been shown previously to recognize both the human and chicken oviduct ER [4]. The genomic organization of the human ER gene was anaalysed by digestion of human (MCF-7) DNA

Cloning of the human ER cDNA

-0.6 Fig. 7. Southern analysis. Human genomic DNA (3Opg)was digested with one of the following enzymes, PvuII, SacI, TaqI, PstI, I&III, HindIII, EcoRI or BamHI and electrophoresed on a 1.5%agarose gel. The DNA was transferred to diazobenzyloxymethyl (DBM) paper and hybridized with the nick-translated insert of dOR8. The position of the DNA size markers are indicated in kilobases.

with several restriction enzymes followed by Southern analysis (Fig. 7). For each digest, AOR hybridized to multiple bands suggesting the organization of the ER gene to be complex. At present we are not certain of the exact copy number of the ER gene, however hybridizations performed using fragments of i-OR8 cDNA give only one or two bands suggesting that the gene may be present as a single copy (data not shown). Therefore, it would appear that approx. 40 kbase (the sum of the fragments in each digest, Fig. 7) of genomic sequence is required to code for 2.1 kbase of cDNA sequence which suggests the existence of long intronic sequences. In contrast, the genomic organization of the chicken gene appears to be less complex, but this may simply reffect a lack of hybridization of some portion of the human cDNA with the chicken gene when hybridized under high-stringency conditions. Hybridization of 10R8 with genomic blots of human-mouse hybrid cell lines have localized the human ER gene to human chromosome 6 (J. L. Mandel: personal communication). DISCWSION

She use of well-characterized monoclonal antibodies against the human ER purified from MCF-7

81

ceils in combination with a cDNA expression library in agtl 1, and of synthetic oligonucleotide probes, derived from amino acid sequences of the purified protein, with a AgtlO cDNA library, has allowed us to isolate cDNA clones corresponding to this receptor. Two of these clones, 10R3, isolated by antibody screening, and /1OR8, isolated using oligonucleotide probes, were able to hybrid select an MCF-7 cell mRNA which could be translated in vitro to yield predominantly a protein which has the same size as the ER (approx. 65 kdalton) and is selectively immunopurified with the ER monoclonal antibodies. Thus the cDNA clones presented here correspond to human ER mRNA. Whenever the in vitro translation products of the MCF-7 poly A+ RNA were examined, either before or after hybrid selection, a weaker additional band of approx. 46 kdalton was observed. The nature of the smaller protein is at present unknown. However, it may correspond to an in vitro degradation product of the larger protein, or to a premature termination of translation, since it was only observed in those denaturing sucrose gradient fractions which yielded the 65 kdalton protein (see Fig. 1). The 46 kdalton component is not observed after immunoprecipitation of iodinated purified MCF-7 ER (unpublished results). The amount of information required to code for a protein of 65 kdalton corresponds to an mRNA of approx. 2 kbase. Since the size of the MCF-7 cell ER mRNA appears to be approx. 6.2 kbase. a large fraction of the mRNA should be untranslated. In the vast majority of eukaryotic mRNAs the most 5’ AUG is used to initiate translation [22]. Therefore the human mRNA is likely to contain a short 5’ and therefore a very long 3’ untranslated region. This would not be unique to the ER mRNA as several other receptor mRNAs appear to have a similar structure [23 to 251. One of the most interesting questions related to steroid hormone research is how does the binding of a hormone to the receptor activate gene expression at the tran~riptional level? It is likely that the ER behaves in a way similar to those of the progesterone and glucocorticoid receptors and that specific promoter elements are in some way responsible for specific gene activation, possibly by directly binding the hormone-receptor complex [26-30, see also reference in 311. Therefore the ER protein may consist of at least two functional domains, the hormone and DNA binding sites, as previously shown f4]. We are at present isolating the chicken ER cDNA using AOR as a probe. Sequence comparison between the human and chicken ER cDNAs, together with in vitro genetics, should allow us to locate these functional domains whose tertiary structure could subsequently be studied. Expression of ER cDNA in transgenic mice under the control of a variety of promoters may help to evaluate the role of ER in regulation of gene expression during development and in terminally differentiated cells. This could be further examined by

STEPHEN GREEN et ul.

82

transfection of an ER cDNA expression vector into cells together with an oestrogen-regulated gene such as pS2 [32]. Similar studies using the ER gene should allow those regions involved in the tissue-specific and developmental regulation of the gene to be localized. Finally, the loss of oestrogen-dependence in some human breast cancers is often associated with the appearance of more malignant tumours [7]. The expression of anti-sense ER mRNA preventing the expression of the endogenous ER in established breast cancer cell lines will offer an opportunity to directly examine the effect of the ER on the growth of these cells. This may lead to a better understanding of the role of the ER in human breast cancer.

12.

13.

14.

15.

16.

Ackno,~,ledgenlent.s~We would like to thank T. Huynh. R. Young and R. Davis of Stanford University for the kind gifts of dgtl0, i.gtl I. associated E. co/i strains and protocols. We are also grateful to Dr L. S. Miller of Abbott Laboratories (Chicago) for providing the H222 and H226 monoclonal antibodies. MCF-7 cells were kindly provided by the Michigan Cancer Foundation. We thank also Mrs B. Heller and L. Heydler for growing the cells and A. Staub for synthesizing the oligonucleotides. This research was supported by INSERM (Grant CNAMTS), the CNRS (ATP 0184) and the Association pour le Developpement de la Recherche sur le Cancer in France, and by the National Cancer Institute (CA07897) and the American Cancer Society (BC86) in the U.S.A. Stephen Green is a recipient of a Royal Society European Exchange fellowship. Colette Goffin is a recipient of a fellowship from the Commission des Communautes Europeenes.

17.

18.

19.

20.

REFERENCES

1. Anderson J. N.: The steroid hormones in gene transcription. In Biological Regulation and Detrelopment (Edited by R. F. Goldberger and K. R. Yamamoto). Plenum Press, New York, Vol. 3B (1984) pp. 169-212. 2. Jensen E. V., Greene G. L., Closs L. E.. DeSombre E. R. and Nadii M.: Receptors reconsidered: a 20-year perspective. In Recenr Progress in Hormone Research (Edited by R. 0. Greep). Academic Press, New York, Vol. 38 (1983) pp. l-34. 3. Greene G. L., Nolan C., Engler J. P. and Jensen E. V.: Monoclonal antibodies to human estrogen receptor. Proc.

natn. Acad.

Sci.

U.S.A.

77 (1980)

5115-5119.

4. Greene G. L.. Sobel N. B., King W. J. and Jensen E. V.: Immunochemical studies of estrogen receptors. 1. steroid Biochem. 20 (1984) 51- 56. antibodies 5. King W. J. and Greene G. L.: Monoclonal localize oestrogen receptor in the nuclei of target cells. Nature

307 (1984)

745.-747.

6. Welshons W. V., Lieberman M. E. and Gorski L.: Nuclear localization of unoccupied oestrogen receptors. Nature

307 (1984)

14lFl49.

7. Henderson I. C. and Canellos G. P.: Cancer of the breast. The past decade. Neiv Enngl. J. Med. 302 (1980) 78-90.

R. A. and Davis R. W.: DNA (Edited by D. Glover). IRL Press. Oxford, Vol. I (1985) pp. 988121. 9. Young R. A. and Davis R. W.: Efficient isolation of genes by using antibody probes. Proc. natn. Acad. Sci. 8. Huynh

Clokng:

U.S.A.

T. V., Young a Practical

80 (1983)

Approach

11941198.

10. Young R. A. and Davis R. W.: Yeast RNA polymerase II genes: isolation with antibody probes. Science 222 (1983) 7788782. 11. Matthes H. W. D.. Zenke W. M., Grunstrom T., Staub A.. Wintzerith M. and Chambon P.: Simultaneous rapid

chemical synthesis of over one hundred oligonucleotides on a micrbscale. EMBO JI 3 (1984) 801-805. Maniatis T., Fritsch E. F. and Sambrook J.: Molecular Cloning: a Laborator? Manual. Cold Spring Harbor Laboratory, Cold Sprmg Harbor, N.Y. (1982). Brooks S. C.. Locke E. R. and Some H. D.: Estroeen receptor in a human cell line (MCF-7) from breast carcinoma. J. hiol. Chem. 248 (1973) 6251-6253. Auffray C. and Rougeon F.: Purification of mouse immunoglobulin heavy-chain messenger RNAs from total myeloma tumor RNA. Eur. J. Bioc/rmr. 107 (I 980) 303.~314. Kraus J. P. and Rosenberg L. E.: Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption. Proc. nafn. Acad. Sci. U.S.A. 79 (1982) 40154019. Long E. O., Gross N.. Wake C. R., Mach J. P.. Carrel S., Accolla R. and Mach B.: Translation and assembly of HLA-DR antigen in Xenopus oocytes injected with mRNA from a human B-cell line. EMBO JI 1 (1982) 6499654. Bailey J. M. and Davidson N.: Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Ana&. Biochem. 70 (1976) 75585. Alwine J. C., Kemp D. J., Parker B. A., Reiser J.. Remart J., Stark G. R. and Wahl G. M.: Detection of specific fragment of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. In Me/ho& in Enzymology (Edited by R. Wu). Academic Press. New York, Vol. 68 (1979) pp. 220-242. LeMeur M., Glanville N.. Mandel J. L.. Gerlinger P., Palmiter R. and Chambon P.: The ovalbumin gene family: hormonal control of X and Y gene transcription and mRNA accumulation. Cell 23 (1981) 561-57‘1. Hoeiismakers J. H.. Borst P., Van den Burg J.. Weissman C. and Cross G. A. M.: The isolati& of plasmids containing DNA complementary to messenger RNA for variant surface glycoproteins of Trypanosomas ”

brucei.

Gene 8 (1980)

391

417.

21. Horwitz K. B.: Is a functional estrogen receptor always required for progesterone receptor induction in breast cancer? J. steroid Biochem. 15 ( 1981) 2099217. and analysis of sequences 22. Kozdk M.: Compilation upstream from the translation start site in eucaryotic mRNAs. Nucleic Acids Res. 12 (1984) 857-872. A. C.. Wrange 0.. 23. Miesfeld R.. Okret S.. Wilkstrom Gustafsson J. A. and Yamamoto K. R.: Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature 312 (1984) 779-781. T.. Davis C. G., Brown M. S., Schneider 24. Yamamoto W. J., Casey M. L., Goldstein J. L. and Russell D. W.: The human LDL receptor: a cystein-rich protein with multiple Alu sequences in its mRNA. Cell 39 (1984) 27738. 25. McClelland A., Kuhn L. C. and Ruddle F. H.: The human transferrin receptor gene: genomic organisation and the complete primary structure of the receptor deduced from a cDNA sequence. CeN 39 (1984) 2677214. 26. Mulvihill E.. LePennec J. P. and Chambon P.: Chicken oviduct progesterone receptor: location of specific regions of hiah affinitv bindine in cloned DNA fraemints of hormone-responsive-genes. CeN 28 (1982) 621~633. 27. Payvar F., Wrange O., Carlstedt-Duke J.. Okret S.. Gustafsson J. A. and Yamamoto K. R.: Purified glucocorticoid receptor binds selectively in vitro to a cloned DNA fragment whose transcription is regulated by glucocorticoids in Lko. Proc. natn. Acud. Sci. U.S.A. 78 (1981) 28.

6628-6632.

Govindan M. V., Spiess E. and Majors _I. E.: Purified glucocorticoid receptor hormone complex from rat liver

Cloning of the human ER cDNA cytosol binds specifically to cloned mouse mammary tumor virus long terminal repeats in oifro. Proc. natn. Acad. S-i. U.S.A. 79 (1982) 5157-5161. 29. Dean D., Gope R., Knoll B. .I., Riser M. E. and

O’Malley B. W.: A similar 5’-flanking region is required for estrogen and progesterone induction of‘ ovalbumin gene expression. J. biof. C.&m. 259 f 1984) 9961-9970. 30. Van der Ahe D., Janich S.. Scheidereit C. and Beato M.: Glucocorticoid and progesterone receptors bind to the same sites in two hormonally regulated promoters. Nawe 313 (1985) 706-709.

SB 24il-c

83

31. Chambon P., Dierich A., Gaub M. P., Jakowlev S., Jongstra J., Krust A., L.ePennec J. P., Oudet P. and Reudelhuber T.: Promoter eIements of proteins and modulation of transcription by estrogens and progesterone. In Recent Progress in Hormone Research (Edited by R. 0. Greep). Academic Press, New York, Vol. 40 (1984) pp. I-42. 32. Masiakowski P., Breathnach R., Block J., Gannon F., Krust A. and Chambon P.: Cloning of cDNA sequences of hormone-regniated genes from the MCF-7 human breast cancer cell line. Nucfeie A&& Res. 10 (1982) 789s-7903.