Poly(A)' RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and fractions enriched in estrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the AgtlO and Agtll vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (Xgtll) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (XgtlO). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, XOR3 (1.3 kilobase pairs) and XOR8 (2.1 kilobase pairs), isolated by using antibodies and oligonudeotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, AOR8 contains the DNA sequence expected from the two ER peptides and crosshybridizes with each of the other ER cDNA dones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of XOR8 as a hybridization probe revealed a single poly(A)+ RNA band of %6.2 kilobase pairs in the ER-containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-negative cell line HeLa. The same probe hybridizes to a chicken gene that is expressed in oviduct tissue as a 7.5-kilobase-pair poly(A)+ RNA.Estrogens, in common with other steroid hormones, regulate gene expression in target cells through their interaction with specific receptors (for review, see ref. 1). The presence of estrogen receptors (ER) can be determined either by their high affinity binding for [3H]estradiol (2) or by using specific monoclonal antibodies (3,4). Recent studies have suggested that the estrogen-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), 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 so far unknown.Further understanding of this mechanism has been severely hampered due to the low level of ER expression. A high level of expression of ER cDNA, in both homologous as well as heterologous systems, should allow further insight into ER structure and function at the molecular level. Since expression of the ER gene is both tissue-specific and developmentally regulated, isolation of the ER gene should lead to the identification of the responsible sequence elements.ER are believed to play an important role in the growth and development of a subset of hormone-dependent human breast cancers. Approximately one-third of all breast cancer tumors contain significant amounts of ER and about twothirds of these are able to respond objectively to some form of anti-est...
Site‐directed mutagenesis was used to prepare a series of human oestrogen receptor (hER) deletion mutants. The ability of these mutant receptors to bind oestradiol, either after being transiently expressed in HeLa cells or produced synthetically in vitro using T7 polymerase coupled with a rabbit reticulocyte lysate translation system, was analysed. The results indicate that a region which is highly conserved (94% amino acid identity) between the human and chicken ERs (region E) contains all of the sequence necessary to bind oestradiol with high affinity. When tight nuclear association of the oestradiol‐receptor complex was investigated using the oestradiol‐binding mutants of the same series, two regions of the hER sequence were found to be important. One of these regions is completely conserved (100% amino acid identity) between the human and chicken ERs (region C). This region is rich in cysteine and basic amino acids and contains motifs similar to those which have been proposed to be important for DNA binding in other eukaryotic transcriptional regulatory proteins. The other region (region D), which is comparatively poorly conserved (38% amino acid identity), is located between the putative DNA‐binding domain (region C) and the oestradiol‐binding domain (region E).
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