VASOACTIVE INTESTINAL POLYPEPTIDE FROM GENE TO PEPTIDE

本项目将结合联合收割机的分子遗传学方法， 血管活性肠肽递质的神经元表达 多肽（VIP）。 我们将尝试克隆VIP的互补DNA（cDNA） 细菌中的mRNA，并使用克隆的cDNA来鉴定“VIP前体”。" cDNA的核苷酸测序将用于鉴定VIP和相关的 肽，其可以存在于相同的前体中。 为了克隆VIP cDNA，我们 使用合成的寡脱氧核苷酸， 从VIP氨基酸序列预测的序列，和来自 大鼠脑产生可能的VIP特异性cDNA，采用酶 逆转录酶 克隆的cDNA将用于鉴定可能的 VIP前体通过与VIP mRNA选择性杂交， 体外翻译 “VIP前体”将通过单克隆抗体检测 和多克隆抗VIP抗体。 然后，我们打算使用克隆的cDNA 材料，以确定编码VIP的基因片段，并试图 克隆并测序这个基因。 这些研究将揭示 基因DNA和转录过程中可能的RNA剪接机制。 到 监测VIP基因在神经系统和内分泌细胞中的表达 在发育、衰老和高血压期间，我们将使用克隆的cDNA 或者通过凝胶印迹和杂交定量VIP mRNA水平 实验或通过原位杂交方法来定位含有VIP的 结构. 此外，抗VIP抗体将用于 免疫组织化学或放射免疫测定。 的意义 建议的研究在于它可能对理解 VIP合成的调节和可能发现的额外的 与VIP共合成的肽或调节分子， 先驱 这些新的调节肽可能在不同的VIP中存在差异。 含有细胞，并可赋予VIP局灶活性的特异性。 最后，这些研究将提供一系列关于 VIP的遗传调节，以前没有为大多数人提供 脑肽

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.