Cloning of the human oestrogen receptor cDNA

Abstract

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 λgt10 and λgt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (λgt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (λgt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, λOR3 (1.3 kbase) and λOR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore,λOR8 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 λOR8 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.