Regulation of ribonucleotide reductase in Aedes aegypti

描述（由申请人提供）：核糖核苷酸还原酶（RNR）催化脱氧核糖核苷酸的从头合成。第一类RNRs是具有同型二聚体的alpha2Beta2全酶，用Ri和R2表示。RI含有活性和变构结合位点；R2含有形成催化必需的酪氨酸自由基所需的双核铁中心。R1和R2的相互作用是酶活性的必要条件。在哺乳动物细胞中，R1和R2的表达在细胞周期的S期达到峰值，并在紫外线照射下上调，从而需要DNA修复。R1和R2的信息与蛋白质水平相关，并且在哺乳动物和酵母中受细胞分裂期间上调的因子的转录调节。目前，人们对这种重要的酶在昆虫体内是如何调节的知之甚少。我们的初步数据表明，RI和R2表达的转录控制发生在黄热病蚊吸血埃及伊蚊之后。我们的建议的中心假设是，一组组织特异性效应由供血诱导的卵发生决定了R1和R2基因的转录调控，这些因子通过抑制或诱导这些基因的启动子活性起作用。这个应用程序的目的是解剖的转录调控RNR以下血液喂养。为了实现这一应用的目标，我们将追求三个具体目标：1)检查血餐诱导R1和R2。我们将评估供血对组织特异性诱导R1和R2信息和蛋白水平的影响。此外，我们将测定喂血后R1和R2基因的最大表达时间。2)获取和定位R1和R2基因的基础启动子。我们将获得R1和R2的基因组克隆。一旦基因组克隆获得，这两个基因的基础启动子将使用瞬时转染测定和dna酶I足迹绘制。3)转录因子的部分鉴定。我们将从具有可诱导R1和R2基因的组织中生成组织特异性表达文库，并使用R1和R2启动子作为基础转录因子的诱饵。然后，我们将使用基础因子来捕获血粉诱导因子。

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.