Regulation of ribonucleotide reductase in Aedes aegypti

性状（由申请方提供）：核糖核苷酸还原酶（RNR）催化脱氧核糖核苷酸的从头合成。I类RNR是α 2 β 2全酶，其同源二聚体表示为R1和R2。R1含有活性和变构结合位点; R2含有形成催化必需的酪氨酰基自由基所需的双核铁中心。R1与R2的相互作用对于酶活性是必需的。在哺乳动物细胞中，R1和R2的表达在细胞周期的S期是最大的，并通过紫外线辐射上调，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由细胞分裂期间上调的因子进行转录调节。目前，人们对这种必需酶在昆虫中是如何调节的知之甚少。我们的初步数据表明，转录控制RI和R2的表达发生后，埃及伊蚊的黄热病蚊子吸血。我们的建议的中心假设是，一组组织特异性效应器诱导的血液喂养卵子决定R1和R2基因的转录调控，这些因素的工作通过抑制或诱导这些基因的启动子活性。本申请的目的是剖析血液喂养后的转录调节RNR。为了实现本申请的目的，我们将追求三个具体目标：1）检查R1和R2的血餐诱导。我们将评估血液喂养对R1和R2信息和蛋白水平的组织特异性诱导的影响。此外，我们还将确定吸血后R1和R2基因最大表达的时间。2)获得R1和R2基因的基础启动子并定位。我们将获得R1和R2的基因组克隆。一旦获得基因组克隆，将使用瞬时转染测定和DNA酶I足迹法定位两种基因的基础启动子。3)转录因子的部分鉴定。我们将从具有诱导型R1和R2基因的组织中产生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餐诱导因子。

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.