Cloning of the human estrogen receptor cDNA.

Abstract

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...

Expression of genes coding for pS2, c‐erbB2, estrogen receptor and the H23 breast tumor‐associated antigen

“…Plasmids containing inserts of ER and pS2 coding sequences were kindly provided by P. Chambon [16,17]. The plasmid recombinant for the erbB2 gene was constructed by T. Yamamoto [IS] and kindly provided by Y. Shilo.…”

Aging and substitutive hormonal therapy influence in regional and subcellular distribution of ERα in female rat brain

“…Estrogen receptor alpha (ERα), the first to be discovered and cloned (Jensen 1962;Walter et al 1985), is a 66-Kda protein in its most common form; estrogen receptor beta (ERβ) was discovered a decade later (Mosselman et al 1996) but both of them present different splicing variants as well as natural occurring mutants.…”

Evaluation of in vitro fertilization parameters and estrogen receptor alpha gene polymorphisms for women with unexplained infertility

“…Two subtypes of estrogen receptors exist in humans; ERα [4] and ERβ [5], coded by ESR1 and ESR2 genes, respectively. ERα and ERβ are both members of nuclear hormone receptor family.…”