Regulation of ribonucleotide reductase in Aedes aegypti

描述(申请人提供)：核糖核苷酸还原酶(RNR)催化脱氧核糖核苷酸从头合成。I类RNRs是含有同源二聚体的α2Beta2全酶，表示为Ri和R2。RI含有活性和变构结合部位；R2含有形成催化必需的酪氨酸基所需的双核铁中心。R1与R2的相互作用是酶活性所必需的。在哺乳动物细胞中，R1和R2的表达在细胞周期的S期达到最大值，并在紫外线照射下上调，需要DNA修复。R1和R2的信息与蛋白质水平相关，在哺乳动物和酵母中受细胞分裂过程中上调的因子转录调控。目前，对昆虫体内这种重要的酶是如何调节的知之甚少。我们的初步数据表明，在黄热病蚊子埃及伊蚊的吸血后，RI和R2的表达发生了转录控制。我们的建议的中心假设是，为了卵子发生而供血诱导的一组组织特异性效应器决定了R1和R2基因的转录调控，并且这些因子通过抑制或诱导这些基因的启动子活性来发挥作用。本应用的目的是剖析血液喂养后RNR的转录调控。为了实现这一应用的目标，我们将追求三个具体目标：1)检测R1和R2的血餐诱导。我们将评估血液喂养对组织特异性诱导R1和R2消息和蛋白水平的影响。此外，我们还将确定血液喂养后R1和R2基因的最大表达时间。2)获取和定位R1和R2基因的基本启动子。我们将获得R1和R2的基因组克隆。一旦获得基因组克隆，两个基因的基本启动子将通过瞬时转基因分析和DNase I足印进行定位。3)转录因子的部分鉴定。我们将从组织(S)中构建具有可诱导的R1和R2基因的组织特异性表达文库，并将R1和R2启动子用作基本转录因子的诱饵。然后，我们将使用基本因素来捕获血餐诱导因素。

DESCRIPTION (provided by the applicant): Ribonucleotide reductase (RNR) catalyzes the de novo synthesis of deoxyribonucleotides. Class I RNRs are alpha2Beta2 holoenzymes with homodimers denoted as Ri and R2. RI contains the active and allosteric binding sites; R2 contains a di-nuclear iron center required for formation of a catalytically essential tyrosyl radical. Interaction of R1 with R2 is mandatory for enzymatic activity. In mammalian cells, R1 and R2 expression is maximal during S phase of the cell cycle and is up regulated by ultraviolet radiation necessitating DNA repair. The messages for R1 and R2 correlate with the protein levels and are transcriptionally regulated in mammals and yeast by factors that are up regulated during cell division. Currently, little is known about how this essential enzyme is regulated in insects. Our preliminary data indicate that transcriptional control of RI and R2 expression occurs following blood feeding of the yellow fever mosquito Aedes aegypti. The central hypothesis of our proposal is that a set of tissue-specific effectors induced by blood feeding for oogenesis dictate the transcriptional regulation of R1 and R2 genes, and that these factors work by repressing or inducing promotor activity of these genes. The objective of this application is to dissect the transcriptional regulation RNR following blood feeding. To accomplish the objective of this application, we will pursue three specific aims: 1) To examine blood meal induction of R1 and R2. We will evaluate the effect of blood feeding on tissue-specific induction of R1 and R2 message and protein levels. In addition, we will determine the time of maximal expression of R1 and R2 genes following blood feeding. 2) To acquire and map the basal promotors of R1 and R2 genes. We will acquire genomic clones for both R1 and R2. Once the genomic clones are obtained, the basal promoters of both genes will be mapped using transient transfection assays and DNase I foot printing. 3) Partial identification of transcriptional factors. We will generate tissue-specific expression library (ies) from tissue(s) with inducible R1 and R2 genes, and use the R1 and R2 promoters as baits for basal transcriptional factors. We will then use the basal factors to trap the blood-meal inducible factors.