STUDY OF THE BIOSYNTHESIS AND GENE EXPRESSION OF NEUROPEPTIDE Y: A NEURONAL HORMONE

CAROLYN ANN DIETER MINTH, Purdue University

Abstract

The polypeptide hormone neuropeptide Y (NPY) is synthesized in the central and peripheral nervous system as well as the endocrine cells of the adrenal medulla. The biosynthesis of NPY was studied by isolating and characterizing the cDNA for NPY. Total RNA was isolated from a human pheochromocytoma, a tumor of the adrenal medulla which overproduces NPY. Double-stranded, dC-tailed cDNA was synthesized and annealed to PstI restriction enzyme digested, dG-tailed pUC8. This mixture was used to transform Escherichia coli JM83. The resulting transformants were screened with a mixture of tetradecamer probes. The probe sequences were based on the known amino acid sequence of porcine NPY. The sequence of the cDNA was determined and found to code for a precursor 97 amino acids in length. The cDNA was used as a probe to isolate the human NPY gene. This gene contains 4 exons and spans approximately 10kb. The exons, the intron-exon junctions, and 5' and 3' flanking sequences were analyzed using the dideoxy sequencing method of Sanger. In order to study the expression of this gene, 581bp of the 5' nontranscribed sequences of the gene including 51bp of exon 1 were subcloned in front of the bacterial gene for chloramphenicol acetyltransferase (CAT). This chimeric plasmid was introduced into CA-77, PC12, and HeLa cell lines and transient expression of the NPY promoter was measured via activity of the CAT gene. These sequences were sufficient for expression of the NPY promoter in the CA-77 and PC12 cell lines, both of which are derived from tissues of neural crest origin. However, these sequences were not sufficient for expression of the NPY promoter in HeLa cells, a cell line of epidermal origin. Additional studies are necessary to delineate those regions of the NPY promoter which are important for tissue specific expression.

Degree

Ph.D.

Subject Area

Molecular biology

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