THE DROSOPHILA CNS: A MODEL FOR PCB NEUROXICITY

果蝇中枢神经系统：PCB 神经毒性模型

• 批准号: 6178775
• 负责人: DERVLA M MELLERICK-DRESSLER
• 金额: $ 7.59万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6178775
• 关键词: DNA binding protein; Drosophilidae; binding sites; biological models; central nervous system; developmental neurobiology; embryo /fetus; epidermal growth factor; gene expression; genetic enhancer element; genetic mapping; genetic transcription; genetically modified animals; growth factor receptors; halobiphenyl /halotriphenyl compound; homeobox genes; invertebrate embryology; mutant; neurotoxicology; nucleic acid sequence; phosphorylation; posttranslational modifications; protein sequence; reporter genes; tissue /cell culture; transcription factor

Cells of both invertebrate and vertebrate embryos that give rise to neurons must correctly receive and interpret positional information throughout development so that terminally differentiated neurons are precisely determined. The mis-interpretation of positional cues can lead to severe developmental abnormalities and in the most extreme case death. Ventral nervous system defective (vnd), an NK-2 type homeobox gene, specifies the identity of midline proximal ventral neuroectodermal cells and neuroblasts in Drosophila. Since the vertebrate homologues of this gene are expressed in parallel domains in the vertebrate CNS, this suggests both regulatory and functional conservation. The overall goal of this proposal is to understand how the Drosophila vnd gene integrates positional information at the transcriptional and post-translational level so that it fits into the hierarchical network involved in CNS dorsal-ventral specification. Specifically, we want to localize the enhancers responsible for vnd expression in transgenic embryos, identify conserved sequence islands in the vnd regulatory domain from two related Drosophila species, perform DNA binding studies with regulators that bind vnd enhancers and confirm the functional significance of the DNA binding sites by mutating the relevant DNA binding sites and testing their activity in transgenic embryos. The epidermal growth factor (EGF) pathway also patterns the developing CNS along the dorsal-ventral axis by inducing the phosphorylation of largely unidentified target proteins. Since expression of Vnd is affected in EGF receptor mutants, we believe that Vnd is regulated by phosphorylation. We want to detemine if Vnd is phosphorylated, and to identify the amino acids that are modified, with the long term goal of determining the role of phosphorylation in Vnd's function as a transcription factor. Finally, we want to address the role of the NK-2 box, a conserved stretch of 18 amino acids downstream of the homeodomain, in Vnd's role as a transcription factor. The capacity of mutated Vnd protein, lacking the NK-2 box, to drive reporter expression will be assayed in a tissue culture model. We recently showed that over-expression of wild-type Vnd leads to a transformation in neuronal precursor identity. Transgenic embryos that over-express mutant Vnd, lacking the NK-2 box, will be assayed for alteration in neuronal fate. These studies directly address the structure and function of vnd, a critical regulator of early neuronal development, and may serve as a prototype for vertebrate vnd-like genes.

产生神经元的无脊椎动物和脊椎动物胚胎细胞必须在整个发育过程中正确地接收和解释位置信息，以便精确地确定最终分化的神经元。对位置线索的错误解释会导致严重的发育异常，在最极端的情况下会导致死亡。腹侧神经系统缺陷（vnd）是一种NK-2型同源盒基因，指定了果蝇中线近侧腹侧神经外胚层细胞和神经母细胞的身份。由于该基因的脊椎动物同源物在脊椎动物中枢神经系统的平行结构域中表达，这表明调控和功能守恒。本研究的总体目标是了解果蝇vnd基因如何在转录和翻译后水平整合位置信息，从而适应中枢神经系统背侧-腹侧规范的分层网络。具体而言，我们希望定位转基因胚胎中负责vnd表达的增强子，从两个相关的果蝇物种中鉴定vnd调控域的保守序列岛，与结合vnd增强子的调控子进行DNA结合研究，并通过突变相关的DNA结合位点并测试其在转基因胚胎中的活性来确认DNA结合位点的功能意义。表皮生长因子（EGF）通路也通过诱导大部分未知靶蛋白的磷酸化，沿背腹轴调控发育中的中枢神经系统。由于Vnd的表达在EGF受体突变体中受到影响，我们认为Vnd受磷酸化调节。我们想要确定Vnd是否被磷酸化，并确定被修饰的氨基酸，长期目标是确定磷酸化在Vnd作为转录因子的功能中的作用。最后，我们想要解决NK-2盒子的作用，这是同源结构域下游18个氨基酸的保守延伸，在Vnd作为转录因子的作用中。缺乏NK-2盒子的突变Vnd蛋白驱动报告基因表达的能力将在组织培养模型中进行检测。我们最近发现野生型Vnd的过度表达导致神经元前体身份的转变。过度表达缺乏NK-2盒子的突变Vnd的转基因胚胎将被用于检测神经元命运的改变。这些研究直接解决了vnd的结构和功能，vnd是早期神经元发育的关键调节因子，并可能作为脊椎动物vnd样基因的原型。

项目成果

Metabolism of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) by liver microsomes and isolated rabbit cytochrome P450 isozymes.

肝微粒体和分离的兔细胞色素 P450 同工酶对 2-氨基-1-甲基-6-苯基咪唑并[4,5-b]吡啶 (PhIP) 的代谢。

• DOI: 10.1093/carcin/11.6.941
• 发表时间: 1990
• 期刊: Carcinogenesis
• 影响因子: 4.7
• 作者: Turteltaub,KW;Knize,MG;Buonarati,MH;McManus,ME;Veronese,ME;Mazrimas,JA;Felton,JS
• 通讯作者: Felton,JS