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  • Title: [Sequencing and cloning of human estrogen beta receptor (hERbeta) cDNA in human granulosa].
    Author: Huang H, Mershon JL, Wang J.
    Journal: Zhonghua Yi Xue Za Zhi; 2000 Jan; 80(1):28-30. PubMed ID: 11798733.
    Abstract:
    OBJECTIVE: To analyze the nucleotide sequence of cDNA and deduce the amino acid sequence of human estrogen receptor (hERbeta) in human granulosa cells. METHODS: 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. RESULTS: 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). CONCLUSION: Detection of ERbeta in the ovary granulosa cells played an important role in explaining the self-endocrine function of estrogen.
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