Primates and equidae are the only mammals known to synthesize chorionic gonadotropin (CG). Although much is known about the molecular events underlying placenta-specific expression of the human alpha subunit gene, little is known about the mechanisms that regulate expression of the closely related human LHbeta and CGbeta genes. Understanding of the mechanisms governing expression of the equine CG genes lags even further behind as these genes that have yet to be cloned and fully characterized. Further study of the equine CG genes is warranted because there are major differences between primates and horses regarding the biology of their pituitary and placental gonadotropins including: biology of placentation, structural and functional properties of CG, structural relationships between LH and CG, mechanisms underlying placenta-specific expression of the alpha subunit gene, and hormonally regulated secretion of pituitary LH. To study the mechanisms underlying tissue-specific expression and hormonal regulation of genes encoding eLH/CG we propose three Specific Aims: 1) define the cis-acting elements required for placenta-specific expression of the horse alpha subunit gene; 2) identify regulatory elements required for hormonal regulation of the eLH/CGbeta gene; and 3) construct transgenic mice and immortalized cell lines for physiological studies on tissue- specific expression and hormonal regulation of the eLH/CG genes. A variety of approaches will be used including: PCR cloning, DNA-protein analyses, transfection of primary cultures of horse placental cells, recognition site cloning, and expression of chimeric genes in transgenic mice. We postulate that a complex and diverse array of cis-acting elements and trans-acting factors will confer tissue-specific expression and hormonal regulation to the horse LH/CG genes. Many of these elements and factors will likely be different than those required for regulated expression of the human CG genes. Furthermore, many of these regulatory elements are probably used by other genes whose expression is targeted to pituitary or placental. Our goal is to develop a complete physiological model to evaluate the biological significance of the large number of tightly packed cis-acting elements found in the promoter region of the gonadotropin genes and their cognate trans-acting factors. We will approach this goal not only by identifying promoter-regulatory elements, but also by developing, simultaneously, a transgenic animal model which can be used to evaluate their physiological significance.
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