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|Title:||Estrogen Receptor Regulation of Xenopus Laevis Vitellogenin Gene Expression|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The mechanism by which the Xenopus laevis estrogen receptor mediates the transcriptional response of the vitellogenin gene was investigated. The Xenopus estrogen receptor (XER) cDNA was cloned and sequenced. It could encode a protein of 66 Kd, which is approximately the same size as human and chicken estrogen receptors. Most of the amino acid homology among these three organisms is concentrated in two regions of the Xenopus ER protein.
A homologous assay system with which to test functional domains in the estrogen receptor protein was developed. The XER cDNA was placed under the control of the herpes simplex virus thymidine kinase promoter (tk), a general constitutive eukaryotic promoter, in order to facilitate its expression in cultured Xenopus fibroblast cells. Xenopus fibroblasts, which do not express endogenous estrogen receptor, were cotransfected with two plasmids: the tkXER cDNA expression plasmid, and a plasmid containing the estrogen-regulated Xenopus vitellogenin gene promoter directing expression of the chloramphenicol acetyl transferase gene (CAT). Only when the two plasmids were cotransfected was CAT activity placed under the control of estrogen in the fibroblasts.
Deletion mutations of the estrogen receptor were constructed in the coding region of the tkXER cDNA expression plasmid and then tested in the cotransfection assay. Deletion of the region of the receptor responsible for binding of estrogen releases vitellogenin transcription from estrogen control. A deletion mutant containing only amino acids involved in DNA binding is still able to activate transcription, but at reduced levels. Interestingly, the mutant containing the DNA binding domain plus the 15 amino acids adjacent to its C terminal side activates transcription almost twice as well as the mutant possessing the DNA binding region alone. These additional residues have the highest concentration of acidic amino acids in the protein.
These mutants delineate the functionally conserved DNA binding and hormone binding domains of the Xenopus estrogen receptor. This study also provides evidence for a conserved acidic region involved in positive transcriptional activation. This information has been useful in helping to elucidate the mechanism of a trans-acting factor, estrogen receptor, in the regulation of gene expression.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
|Date Available in IDEALS:||2014-12-15|