The long-term goal of the present application is the elucidation of the effects of estrogen on specific protein synthesis in the liver. The experimental model is the estrogen-treated cockerel. The estrogen regulated protein studied is apoVLDL-II, an estrogen induced protein first isolated in our laboratory. Previous studies indicate that a major factor in apoVLDL-II induction is the recruitment of liver cells not previously engaged in its synthesis. Control proteins comprise avian vitellogenin, apoA-1, ovalbumin, glycraldehyde 3 phosphate dehydrogenase and Beta-globin. They are chosen because they are either estrogen responsive, estrogen nonresponsive, or unexpressed in the avian liver and their cDNA clones are available. Isolated avian hepatocytes from untreated or estrogen-treated cockerels will be separated into subpopulations of cells that synthesize apoVLDL-II and those that do not. Techniques for cell separation include; (i) the use of the fluorescence activated cell sorter (FACS), and (ii) specific attachment of apoVLDL-II-synthesizing cells onto antibody immobilized on plastic plates. By these techniques at various times after estrogen, the proportion of hepatocytes which synthesize apoVLDL-II can be quantified accurately, and the relationship of the response to the cell cycle can be examined. The apoVLDL-II gene will be studied in untreated and estrogen-treated animals, and in the subpopulations of hepatocytes separated according to whether they synthesize apoVLDL-II or not. The apoVLDL-II gene will be studied in three ways: (i) its possible preferential enrichment in nuclear matrix, (ii) its DNAase I-hypersensitivity, and (iii) its methylation patterns. Specific nuclear estradiol binding sites will also be characterized in these subpopulations of cells. Furthermore, nuclear matrix isolated from these cells will also be examined for the presence of specific estrogen binding sites. Our approach should generate important new information on the molecular basis of the apoVLDL-II response to estrogen, and possibly the mechanisms underlying the heterogeneity of the response amongst different liver cell subpopulations.
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