[Sequencing and cloning of human estrogen beta receptor (hERbeta) cDNA in human granulosa]

To analyze the nucleotide sequence of cDNA and deduce the amino acid sequence of human estrogen receptor (hERbeta) in human granulosa cells.

Granulosa cells were prepared from the ovary of IVF-ET cases by Percoll technique with Dulbecco's modified eagle medium. RNA was extracted with the TRIzol reagent kit, and mRNA was purified with oligo-(dT)-cellulose in a sterile dispocolumn. cDNA was synthesized by RT-PCR using the SuperScript(TM) II RT kit. Amplified products were cloned into the pGEM-T vector and transfected into E. coli XL1-Blue. The nucleotide sequences were determined by repeated sequencing of both strands of alkaline-denatural plasmid DNA using the Sequenase Version 2.0 DNA sequencing kit. The obtained DNA sequence was compiled and analyzed using DNAMAN computer programs.

Amplified cDNA of hER beta in human granulosa cells was composed of 1 495 bp, containing a 1 431 bp open reading frame. The predicted ER beta protein consisted of 477 amino acids. The predicted ER protein included 4 function domains: A/B, C, D, and E/F domains. Among these domains, C domain, richly containing cysteine, was the DNA-binding domain (DBD), and E/F domain was the ligand-binding domain (LBD).

Detection of ERbeta in the ovary granulosa cells played an important role in explaining the self-endocrine function of estrogen.