The overall goal of this research is to understand the regulated biosynthesis of bovine parathyroid hormone (PTH) and parathyroid secretory protein (PSP) which is cosecreted with PTH. PTH is the principle hormone involved in maintaining calcium homeostasis and the activity of the parathyroid gland is controlled by extracellular calcium in a simple feedback loop. We propose to study structural characteristics of the activated bovine PTH gene, the regulatory function of the 5 feet flanking region of the gene and to analyze structure-activity relationships of the signal sequence of preProPTH. We also propose to initiate studies on the biosynthesis of PSP and the closely related protein, chromogranin A. The extent of methylation of the PTH gene will be assessed by differential sensitivity of the PTH gene to digestion with Hha 1 in parathyroid and non parathyroid DNA. The DNase 1 sensitivity and hypersensitive sites will be assessed by incubation of nuclei from parathyroid and liver with dnase 1 followed by Southern blot analysis. The 5 feet flanking region of the cloned PTH gene and the region of the cloned cDNA that codes for the signal sequence will be altered by in vitro mutagenesis, including deletion mutations, cluster mutations by linker scanning techniques, and point mutations by bisulfite or oligonucleotide site-directed techniques. Transcription of the PTH gene will be assayed by injection into Xenopus oocytes and by transformation of mammalian cells with SV40 or bovine papilloma virus vectors carrying the gene. The function of the signal sequence will be assayed by linked transcription-translation in a reticulocyte cell free system and by in corporation of the cDNA into expression vectors and transformation of E. coli and mammalian cells. To initiate studies on the biosynthesis of PSP, poly(A) RNA will be isolated from porcine adrenal medulla and cDNA synthesized from the RNA will be isolated by molecular cloning. Chromogranin a cDNA will be selected by hybridization to cDNA to parathyroid RNA and by hybrid-selection and translation of mRNA. The sequence of the cloned cDNA will be determined by the chemical method or by dideoxynucleotide termination techniques.
