REGULATION OF A DEVELOPMENTALLY CRITICAL INSECT PROTEIN

建议拨款的目的是利用差额 一种发育调节蛋白AS的调控机制 研究发育的荷尔蒙调节。这种蛋白质， 保幼激素酯酶(JHE)，受制于神经激素 在发展过程中的一次监管，然后通过其 自己的底物。因此，该系统对于一种比较的 研究JH如何调控基因表达，以及JH如何 同样的基因可以由化学上无关的荷尔蒙调节。 因为一些医学上重要的昆虫和其他 无脊椎动物已被证明拥有幼体或对幼体做出反应 荷尔蒙，拟议的研究结果可能会发现新的 控制这些生物体的方法。 拟议研究的具体目的是：1)提供 JH酯酶的发育概况及其电泳谱和 在其合成部位(脂肪体)和在 血淋巴，2)测定N-末端序列和其他 蛋白质的生化特性，3)检测 激素调节JHE的生化水平，4)准备 用于测量细胞变化的蛋白质(S)的cDNA克隆 蛋白质在正常发育和发育过程中的mRNA水平 在荷尔蒙操纵之后。 将使用体外脂肪体和体内程序。 将使用酶和免疫学方法来测量 电泳型和动态型的正常变化 就是这样。电泳型将单独提纯N- 末端测序和生化特性。这个N- 末端序列将用于合成寡核苷酸 探针，用来筛选酶的克隆和 已确认身份。这个探针，以及免疫学和酶学 技术，将用于测试 神经激素因子和保幼激素 生物化学水平的调节：mRNA丰度的变化， 可翻译信使核糖核酸水平的变化，数量的变化 脂肪体中的非活性和活性JHE及其释放速率 脂肪的身体，改变了JH酯酶的周转。

The objective of the proposed grant is to use the differential mechanisms of control of a developmentally regulated protein as study of hormonal regulation of development. The protein, juvenile hormone esterase (JHE), is subject to neurohormonal regulation at one time during development and then later by its own substrate. Thus, the system is attractive for a comparative study on how JH acts to regulate gene expression, and how the same gene can be regulated by chemically unrelated hormones. Since a number of medically important insects and other invertebrates have been shown to possess, or respond to, juvenile hormone, the results of the proposed study may identify new approaches to the control of these organisms. The specific aims of the proposed study are: 1) provide the developmental profile of JH esterase and its electrophoretic and kinetic forms, both in their site of synthesis (fat body) and in the hemolymph, 2) determine the N-terminus sequence and other biochemical characteristics of the protein, 3) test for the biochemical level at which the hormones regulate JHE, 4) prepare cDNA clones for the protein(s) to use in measuring changes in mRNA levels of the protein during normal development and following hormonal manipulation. Both in vitro fat body and in vivo procedures will be used. Enzymatic and immunological methods will be used to measure the normal changes in the electrophoretic and kinetics forms of JHE. The electrophoretic forms will be separately purified for N- terminus sequencing and biochemical characterization. The N- terminus sequence will be used to synthesize an oligonucleotide probe, with which clones for the enzyme will be screened and identified. This probe, and the immunological and enzymatic techniques, will be used to test for the action of the neurohormonal factor, and juvenile hormone, on the following biochemical levels of regulation: changes in mRNA abundance, changes in level of translatable mRNA, change in amount of inactive and active JHE in the fat body and rate of release from the fat body, change in JH esterase turnover.