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METABOLIC REGULATION OF VASOPRESSIN AND OXYTOCIN

加压素和催产素的代谢调节

基本信息

批准号：
3232560
负责人：
JOEL F HABENER
金额：
$ 10.45万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
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：
1984-08-01 至 1989-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3232560
关键词：
bacterial virus chromatography complementary DNA gene expression gene mutation genetic library genetic transcription hormone regulation /control mechanism hypothalamus messenger RNA molecular cloning neurophysins nucleic acid sequence oxytocin physiologic stressor radioassay stress vasopressins

项目摘要

Vasopressin and oxytocin nonapeptides synthesized in the hypothalamus and secreted via axons in the neurohypophysis, regulate water conservation, and smooth muscle contractions. Both peptides are synthesized in the form of precursors in covalent linkages with neurophysin peptides, termed preprovasopressin-neurophysin and preprooxytocinneurophysin. Studies of vasopressin and oxytocin biosynthesis in this proposal center on investigations of two aspects of vasopressin and oxytocin gene expression: (1) investigations of the molecular mechanisms involved in the regulation of gene expression and (2) elucidation of the molecular defect in gene expression manifested in the Brattleboro rat, a strain of rat with an inborn defect in the production of vasopressin. In preliminary studies we have used synthetic oligonucleotides complementary to vasopressin and oxytocin mRNAs to specifically prime the synthesis of cDNAs from hypothalamic RNA of rats. These oligonucleotide probes are also used in hybridization assays to evaluate steady-state cellular levels of the specific vasopressin and oxytocin mRNAs. We propose to study the regulation of the expression of the vasopressin and oxytocin genes in the hypothalamus of rats in vivo as a consequence of osmotic stress and in hypothalami in vitro in response to changes in the osmolality of the incubation media. We will use blot hybridization techniques to measure steady state mRNA levels in the hypothalami in response to the stimuli, attempt to measure gene transcription rates by polymerase-runoff experiments and evaluate rates of RNA turnover by pulse-labeling studies of hypothalami in vitro. We found that the hypothalamus of the Brattleboro rat contains hybridizable RNA encoding prepropressophysin-neurophysin but in much lower amounts than is found in the syngeneic normal rat. We propose to clone and structurally characterize recombinant cDNAs prepared to mRNAs from the hypothalami of Long-Evans and Brattleboro rats by nucleotide sequencing and S-1 nuclease mapping experiments to determine whether there is a defect (base mutation, sequence insertion/deletion). We plan to prepare genomic libraries of recombinant bacteriophages from the DNA of the Brattleboro and Long Evans rats and to select and structurally characterize the vasopressin gene. Our interest in carrying out these studies resides in the fact that the genes encoding vasopressin and oxytocin have a bifunctionality in that they are expressed both as endocrine hormones and as local cell-to-cell neuromodulators or neurotransmitters.

加压素和催产素九肽在下丘脑合成，通过神经垂体中的轴突分泌，调节水分保存，平滑肌收缩两种肽都是以前体与neurophysin肽共价连接，称为前加压素原-神经后叶素和前催产素原神经后叶素。研究加压素和催产素的生物合成，加压素和催产素基因表达的两个方面的研究： (1)参与调节的分子机制的研究基因表达的分子缺陷的阐明 Brattleboro大鼠（一种具有血管加压素产生的先天缺陷。在初步研究中，已经使用了与加压素互补的合成寡核苷酸，催产素mRNA特异性地引发来自大鼠下丘脑RNA。这些寡核苷酸探针也用于杂交测定以评估细胞内的稳态水平，特异性血管加压素和催产素mRNA。我们建议研究垂体后叶素和催产素基因表达的调节大鼠下丘脑在体内作为渗透应激的结果，体外下丘脑对渗透压变化的反应孵育培养基。我们将使用印迹杂交技术来测量下丘脑中响应刺激的稳态mRNA水平，用聚合酶-径流法测定基因转录速率的尝试实验和评估RNA周转率的脉冲标记研究离体下丘脑。我们发现布拉特波罗人的下丘脑大鼠含有编码前垂体后叶素-神经垂体后叶素的可杂交RNA，比同系正常大鼠中的含量低得多。我们建议克隆和结构表征制备的重组cDNA Long-Evans和Brattleboro大鼠下丘脑的mRNA，核苷酸测序和S-1核酸酶作图实验，以确定是否存在缺陷（碱基突变、序列插入/缺失）。我们计划制备重组噬菌体的基因组文库， Brattleboro和Long Evans大鼠的DNA，血管加压素基因的特征。我们在执行这些方面的兴趣研究表明，编码加压素的基因和催产素具有双功能性，因为它们被表达为内分泌激素和作为局部细胞对细胞的神经调节剂，或神经传递素