VASOACTIVE INTESTINAL POLYPEPTIDE FROM GENE TO PEPTIDE

本项目将结合分子遗传学的方法来研究

This project will combine methods of molecular genetics to study the neuronal expression of the peptide transmitter vasoactive intestinal polypeptide (VIP). We will attempt to clone DNA complemental (cDNA) to VIP mRNA in bacteria and use the cloned cDNA to identify "VIP precursor(s)." Nucleotide sequencing of the cDNA will be used to identify VIP and related peptides, which may exist in the same precursor. To clone the VIP cDNA, we use synthetic oligodeoxynucleotides, with relatively unambiguous nucleotide sequence predicted from the VIP amino acid sequence, and poly (A) mRNA from rat brain to generate possible VIP specific cDNA, employing the enzyme reverse transcriptase. The cloned cDNA will be used to identify possible VIP precursor(s) by selective hybridization to VIP mRNA followed by translation in vitro. "VIP precursor(s)" will be detected by monoclonal and polyclonal anti-VIP antibodies. We then intend to use the cloned cDNA material to identify the gene fragment coding for VIP and to attempt to clone and sequence this gene. These studies will reveal the structure of the gene DNA and possible RNA splicing mechanisms during transcription. To monitor VIP-gene expression in the nervous system and in endocrine cells during development, aging and hypertension, we will use the cloned cDNA either to quantitate VIP mRNA levels by gel blotting and hybridization experiments or by in situ hybridization methods to locate VIP containing structures. In addition, anti-VIP antibodies will be used in immunohistochemistry or radioimmunoassays. The significance of the proposed research lies in its possible contributions to the understanding of the regulation of VIP synthesis and the possible discovery of additional peptides or regulatory molecules cosynthesized with VIP on a common precursor. These novel regulatory peptides may differ in the various VIP containing cells, and may confer specificity on VIP focal activity. Finally, these studies will provide a body of information concerning the genetic regulation of VIP that has not previously been available for most brain peptides.