Hyperplasias of the human pituitary are often associated with abnormally high prolactin production. Regulation of prolactin biosynthesis in animal models is diverse and depends on the species studied. In the rat, estrogens are a major regulator of prolactin biosynthesis in the pituitary gland. The rat pituitary can develop tumors in response to estrogens. Such tumors are the source of the widely used cell culture line, GH3 cells. We propose to study special lines of GH3 cells, developed in our laboratory, which contain the regulatory region of the prolactin gene in an episomal plasmid, or minichromosome, derived from the bovine papilloma virus. These mini-chromosomes respond to estrogen treatment by increasing the expression of a reporter gene linked to the prolactin promoter. Furthermore, they are present in approximately 50 copies per cell and thus increase the possibility of detecting subtle changes that are difficult or impossible to observe in the endogenous single-copy prolactin gene. We plan to focus future studies on the estrogen and antiestrogen regulation of chromatin structure of the prolactin regulatory region contained in the minichromosome system. These studies will make use of DNase sensitivity analysis to determine if estrogen is modifying the structure of two specific regions of the chromatin. These two regions of the chromatin are in a nuclease by estrogens and antiestrogens. We also are proposing to study the topological state of the chromatin by directly examining variation in plasmid topoisomers present in estrogen and antiestrogen treated cells. Proteins present in the minichromosomes will be studied by cross linking the proteins to the minichromosomes in intact cells. Protein-nucleic acid adducts can then be studied using specific antibodies to proteins like the estrogen receptor. We also will determine the affinity of specific proteins, such as the estrogen receptor, for specific DNA fragments from the regulatory regions of the prolactin gene.
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