We have cloned and sequenced the complete complementary DNA of the oestrogen receptor (ER) present in the breast cancer cell line MCF-7. The expression of the ER cDNA in HeLa cells produces a protein that has the same relative molecular mass and binds oestradiol with the same affinity as the MCF-7 ER. There is extensive homology between the ER and the erb-A protein of the oncogenic avian erythroblastosis virus.

A chicken oviduct cDNA clone containing the complete open reading frame of the oestrogen receptor (ER) has been isolated and sequenced. The mol. wt of the predicted 589‐amino acid protein is approximately 66 kd which is very close to that of the human ER. Comparison of the human and chicken amino acid sequences shows that 80% of their amino acids are identical. There are three highly conserved regions; the second and third of which probably represent the DNA‐ and hormone‐binding domains of the receptor. The putative DNA‐binding domain is characterised by its high cysteine and basic amino acid content, and the hormone‐binding domain by its overall hydrophobicity. These two domains of homology are also present in the human glucocorticoid receptor (GR) and the product of the avian erythroblastosis virus (AEV) gene, v‐erbA, indicating that c‐erbA, the cellular counterpart of v‐erbA, belongs to a multigene family of transcriptional regulatory proteins which bind steroid‐related ligands. The first highly conserved ER region is not present in the truncated v‐erbA gene, but shares some homology with the N‐terminal end of the GR. The function of the v‐erbA gene product is discussed in relation to its homology with the ER and GR sequences.

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...

Metal ions, and magnesium in particular, are known to be involved in RNA folding by stabilizing secondary and tertiary structures, and, as cofactors, in RNA enzymatic activity. We have conducted a systematic crystallographic analysis of cation binding to the duplex form of the HIV-1 RNA dimerization initiation site for the subtype-A and -B natural sequences. Eleven ions (K+, Pb2+, Mn2+, Ba2+, Ca2+, Cd2+, Sr2+, Zn2+, Co2+, Au3+ and Pt4+) and two hexammines [Co (NH3)6]3+ and [Ru (NH3)6]3+ were found to bind to the DIS duplex structure. Although the two sequences are very similar, strong differences were found in their cation binding properties. Divalent cations bind almost exclusively, as Mg2+, at 'Hoogsteen' sites of guanine residues, with a cation-dependent affinity for each site. Notably, a given cation can have very different affinities for a priori equivalent sites within the same molecule. Surprisingly, none of the two hexammines used were able to efficiently replace hexahydrated magnesium. Instead, [Co (NH3)4]3+ was seen bound by inner-sphere coordination to the RNA. This raises some questions about the practical use of [Co (NH3)6]3+ as a [Mg (H2O)6]2+ mimetic. Also very unexpected was the binding of the small Au3+ cation exactly between the Watson-Crick sites of a G-C base pair after an obligatory deprotonation of N1 of the guanine base. This extensive study of metal ion binding using X-ray crystallography significantly enriches our knowledge on the binding of middleweight or heavy metal ions to RNA, particularly compared with magnesium.