Epidermal growth factor (EGF) is a polypeptide whidh promotes the growth and differentiation of a great variety of cell types. Its in vivo effects in the mouse include the induction of premature eyelid opening and incisor eruption, the maturation of the lung and trachya, and the inhibition of gastric acid secretion. These potent biological activities are also produced by urogastrone, the human analog of mouse EGF. The importance of the appropriate regulation of EGF activity is emphasized by the recent finding that its membrane receptor is homologous to the oncogene, v-crbB. The major source of mouse EGF is the granular convoluted tubule (GCT) cells of the submandibular salivary gland, which also produce nerve growth factor, renin and kallikreins. In this gland, the expression of EGF is under developmental and multihormonal control. Using an EGF cDNA clone, we have found that EGF mRNA is also produced in the kidney, and that developmental and hormonal regulation in this organ is different from that observed in the submandibular gland. The gene encoding mouse EGF appears to be represented by a single copy per haploid genome. The EGF gene can thus serve as a model for studying tyhe differential regulation of the same DNA sequence in two different tissues. The longterm goal of these studies is to identify the DNA sequences within and surrounding the mouse EGF gene that are required for its multihormonal and developmental regulation. We therefore propose: (1) to perform a series of in vivo studies comparing the regulation of EGF expression by thyroid hormones, androgen, estrogen and development in the kidney and submandibular gland; (2) to isolate and characterize the mouse EGF gene; (3) to develop a cell culture system which can be used for studying the regulation of endogenous and transfected EGF genes; and (4) to begin DNA-mediated gene transformation studies designed to identify the regulatory sequences of the EGF gene. These studies will utilize many of the techniques of molecular biology, including: (1) Northern blot, dot blot, and in situ hybridization to measure relative amounts of EGF RNA and identify the cells within each organ which produce it; 2) nuclear run-off assays to measure relative transcriptional rates of the EGF gene; and 3) DNA sequencing, Southern blot hybridization and restriction endonuclease mapping to characterize the structure of the EGF gene.
