: 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.
| Pham, Daphne Q-D; Kos, Peter J; Mayo, Jonathan J et al. (2006) Regulation of the ribonucleotide reductase small subunit (R2) in the yellow fever mosquito, Aedes aegypti. Gene 372:182-90 |
