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VASOACTIVE INTESTINAL POLYPEPTIDE FROM GENE TO PEPTIDE

血管活性肠多肽从基因到肽

基本信息

批准号：
3398974
负责人：
ILLANA GOZES
金额：
$ 3.91万
依托单位：
WEIZMANN INSTITUTE OF SCIENCE
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-01-01 至 1988-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3398974
关键词：
antibody formation complementary DNA gene expression genetic manipulation messenger RNA molecular cloning monoclonal antibody nucleic acid sequence tissue /cell culture

项目摘要

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.

这个项目将结合分子遗传学的方法来研究血管活性肠肽递质的神经元表达多肽(VIP)。我们将尝试克隆与VIP互补的DNA(CDNA) 并用克隆的c DNA鉴定“血管活性肠炎前体(S)”。该基因的核苷酸序列将用于鉴定VIP及其相关基因多肽，可能存在于相同的前体中。为了克隆血管活性肠肽基因，我们使用具有相对明确的核苷酸的合成寡核苷酸从VIP氨基酸序列预测的序列，以及来自利用该酶产生大鼠脑内可能的VIP特异性cDNAs 逆转录酶。克隆的cdna将用于鉴定可能的血管活性肠肽前体(S)与血管活性肠肽mRNA的选择性杂交体外翻译。“贵宾前体(S)”将用单抗进行检测和多克隆抗血管活性肠肽抗体。然后我们打算使用克隆的cdna 材料鉴定编码VIP的基因片段并尝试对这个基因进行克隆和测序。这些研究将揭示转录过程中基因DNA和可能的RNA剪接机制。至监测VIP基因在神经系统和内分泌细胞中的表达在发育、衰老和高血压过程中，我们将使用克隆的cdna 或者通过凝胶印迹和杂交来定量VIP mRNA的表达实验或通过原位杂交方法定位VIP 结构。此外，抗VIP抗体将用于免疫组织化学或放射免疫分析。这一事件的意义提出的研究在于其对理解的可能贡献对VIP合成的调节以及可能发现的额外的与VIP共同合成的多肽或调节分子前身。这些新的调节肽可能在不同的VIP中有所不同含有细胞，并可能赋予VIP焦点活性的特异性。最后，这些研究将提供关于对VIP的遗传调控，这是以前大多数人都没有的脑多肽。