Cloning of the Human Estrogen Receptor cDNA

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 λ gt10 and λ gt11 vectors. Clones corresponding to ER sequence were isolated from both libraries after screening with either ER monoclonal antibodies (λ gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (λ gt10). Five cDNA clones were isolated by antibody screening and five were isolated after screening with synthetic oligonucleotides. The two largest ER cDNA clones, λ OR3 (1.3 kilobase pairs) and λ OR8 (2.1 kilobase pairs), isolated by using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, λ OR8 contains the DNA sequence expected from the two ER peptides and cross-hybridizes with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Use of λ OR8 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.