The present proposal addresses the problem of gene regulation during development. The purpose of these studies will be to identify regulatory sequences in the 5' promoter/flanking regions of the human gonadotropin subunit genes which control the temporal, tissue and cell type specific expression of these genes during development. The genes to be studied wil include the human gonadotropin alpha subunit gene, expressed in the anterior pituitary (AP) and placenta, the human LH beta gene, expressed in the AP, and the HCG beta 3 gene, expressed in the placenta. The short term goal of these studies will be to construct fusion genes between the promoterless chloramphenicol acetyltransferase (CAT) gene and each of the 5' promoter/flanking regions of the gonadotropin subunit genes. Endonuclease digestion and/or digestion with Bal 31 will be used to generate gonadotropin-CAT fusion genes with reduced 5' flanking sequences. Mice transgenic for the intact and deleted fusion genes will be generated and the tissue specific expression of the different constructs will be examined by assaying for CAT enzyme activity in the AP and other tissues. To determine whether appropriate cell type specific and temporal expression of these genes occurs during development, the 5' promoter/flanking regions retaining tissue specificity by analysis of CAT constructs above will be used to promote expression of the bacterial enzyme beta-galactosidase (beta-gal). Expression of gonadotropin-beta-gal fusion genes will be examined at different stages of development in transgenic mice by histochemical localization of beta-gal at the cellular level. Mice transgenic for gonadotropin-CAT fusion genes will also be used to identify sequences involved in the hormonal regulation of the gonadotropin genes in the AP. Several factors including GnRH, EGF, PMA, cAMP and steroids wil be tested for their ability to influence expression of selected fusion constructs in transgenic APs explanted into culture. The long term goal of these studies will be to analyze in more detail by sequencing and, where possible, additional deletion analysis, those DNA regions found to be important for the developmental and/or hormonal regulation of the gonadotropin subunit genes.
| Fox, N; Solter, D (1988) Expression and regulation of the pituitary- and placenta-specific human glycoprotein hormone alpha-subunit gene is restricted to the pituitary in transgenic mice. Mol Cell Biol 8:5470-6 |
