CONTROLLING THE ACTIVITY OF DROSOPHILA TGF-BETA HOMOLOG

描述：迈克尔·奥康纳博士的续签申请 描述研究发育信号系统的实验。 在果蝇身上。这个系统中的一个关键元素是 Decapentaplegic(DPP)基因编码属于 转化生长因子-β家族。在果蝇早期胚胎中，这是 蛋白质作为控制背腹方向图案的信号 轴，在后来的发展中，它有助于诱导形成 纸巾。脊椎动物中的同源蛋白质与骨骼有关 形态发生。 DPP蛋白作为一种配体刺激细胞膜活性- 果蝇细胞中的结合受体。它可能会协同完成这项工作 另一种类似转化生长因子β的蛋白是螺旋(SCW)基因的产物， 在被Tolloid编码的金属蛋白酶(TLD)处理后 吉恩。另一种由短肠原肠形成(Sog)基因编码的蛋白质可能 拮抗DPP功能。两种不同类型的转化生长因子-β受体 在其他生物体中也被发现。II型受体似乎是 配体结合的主要试剂，一旦结合，似乎会招募 I型受体并激活它们。在果蝇体内，蛋白质 由萨克斯管(Sax)编码的粗脉(Tkv)和atr-i基因出现。 作为I型受体，由Punt基因编码的蛋白质似乎 作为一种II型受体。在这些中，除了ATR-I之外，所有的都被研究过 从基因上来说。 奥康纳博士研究计划的第一部分描述了 对TLD蛋白的功能进行分析。具体目标有五个： (1)确定TLD是否作为酶原分泌；(2)确定是否 TLD蛋白的调节加工对其功能很重要 在体内；(3)通过错误表达来分析TLD的功能 有条件激活的转基因；(4)TLD蛋白在胚胎中的定位 使用表位标记分子的免疫标记或TLD与 水母绿色荧光蛋白；和(5)检测物理 TLD、DPP、Scw和SOG蛋白之间的相互作用。 奥康纳博士研究计划的第二部分概述了 研究果蝇体内的转化生长因子-β受体。有九个具体的 目的：(1)确定PINT基因零突变的表型； (2)获得ATR-I基因突变；(3)使用截短受体 具有轻微显性负效应的转基因作为遗传基础 筛选增强这些影响的突变，以便识别 DPP信号通路的其他组件；(4)使用 成分活性、配基无关的受体，占主导地位 负效应作为基因筛查突变的基础 抑制这些作用；(5)在酵母中使用激活的受体两个- 杂交筛选与蛋白激酶结构域相互作用的蛋白质 Tkv和Sax受体；(6)使用成分激活的受体作为 探讨DPP信号转导潜力；(7)研究SOG模型 蛋白质功能；(8)构建嵌合型I型受体以 研究配体结合特性和激酶的程度 这些受体的活性调节DPP的不同方面 信令；以及(9)构建I型和II型异构体 受体来确定庞特蛋白的体内合作伙伴。

DESCRIPTION: This continuation application from Dr. Michael O'Connor describes experiments to investigate a system of developmental signalling in Drosophila melanogaster. A critical element in this system is the decapentaplegic (dpp) gene, which encodes a protein that belongs to the TGF-beta family of growth factors. In early Drosophila embryos, this protein acts as a signal to control patterning along the dorsal-ventral axis, and later in development, it helps to induce the formation of tissues. Homologous proteins in vertebrates are involved in bone morphogenesis. The Dpp protein acts as a ligand to stimulate the activity of membrane- bound receptors in Drosophila cells. It may do this in concert with another TGF- beta-like protein, the product of the screw (scw) gene, after being processed by a metalloprotease encoded by the tolloid (tld) gene. Another protein encoded by the short gastrulation (sog) gene may antagonize Dpp function. Two different types of TGF-beta receptors have been identified in other organisms. The Type II receptors appear to be the primary agents of ligand binding, and once bound, seem to recruit the Type I receptors and activate them. In Drosophila, the proteins encoded by the saxaphone (sax), thick veins (tkv) and atr-I genes appear to be Type I receptors and the protein encoded by the punt gene seems to be a Type II receptor. Of these, all but atr-I have been studied genetically. The first part of Dr. O'Connor's research plan describes experiments to analyze the function of the Tld protein. There are five specific aims: (1) to determine if Tld is secreted as a zymogen; (2) to ascertain if regulated processing of the Tld protein is important for its function in vivo; (3) to analyze Tld function by misexpressing it through conditionally active transgenes; (4) to localize Tld protein in embryos using immunolabeling of epitope-tagged molecules or Tld fusions with the jellyfish green flourescent protein; and (5) to test for physical interactions among the Tld, Dpp, Scw and Sog proteins. The second part of Dr. O'Connor's research plan outlines experiments to study the TGF-beta receptors in Drosophila. There are nine specific aims: (1) to determine the phenotype of null mutations in the punt gene; (2) to obtain mutations in the atr-I gene; (3) to use truncated receptor transgenes with slight dominant negative effects as the basis of genetic screens for mutations that enhance these effects in order to identify other components of the Dpp signalling pathway; (4) to use constitutively active, ligand independent receptors with dominant negative effects as the basis of genetic screens for mutations that suppress these effects; (5) to use activated receptors in a yeast two- hybrid screen for proteins that interact with the kinase domains of the Tkv and Sax receptors; (6) to use constitutively activated receptors as probes of Dpp signalling potential; (7) to investigate models of Sog protein function; (8) to construct chimeric Type I receptors to investigate the degree to which the ligand binding properties and kinase activities of these receptors mediate different aspects of Dpp signalling; and (9) to construct heterochimeric Type I and Type II receptors to determine the in vivo partner of the Punt protein.