The aim of this study was to determine whether functional ecdysone response elements (EcREs) exist within the genome of Brugia malayi, a parasitic nematode that causes lymphatic filariasis. The hypothesis that EcREs exist in B. malayi stemmed from previous demonstration of a functional ecdysone response system in B. malayi (Tzertzinis et al., 2010). Real-time PCR (qPCR) experiments were conducted to measure gene expression levels for twelve genes proximal to five putative EcREs in 20-OH ecdysone treated and untreated B. malayi embryos. Seven genes showed consistent upregulation with 20-OH ecdysone treatment. Each of the five putative EcREs had at least one proximal gene consistently upregulated, suggesting that all five might be functional EcREs. One of the genes consistently upregulated in the qPCR experiments, Bm1_48650, codes for a MIZ zinc finger family protein, a likely transcription factor. Transgenic ecdysone induction assay experiments were conducted using embryos transiently transfected with a reporter construct driven by the EcRE-containing promoter of Bm1_48650. Significantly higher mean reporter gene activity (~3.5-fold) was seen in 20-OH ecdysone treated versus untreated embryos. In another set of transgenic ecdysone induction assays, the EcRE motif in the Bm1_48650 promoter was completely mutated, and this construct was tested in 20-OH ecdysone treated and untreated embryos. The mean reporter gene activity for the treated and untreated embryos transfected with the mutant constructs did not differ significantly from the untreated embryos transfected with the native EcREcontaining promoter construct. These results showed that the EcRE in the promoter of Bm1_48650 is necessary for regulating gene expression in response to 20-OH ecdysone. This study substantiates previously discovered evidence of a functional ecdysone response system in B. malayi, which could potentially serve as a target for drug discovery for lymphatic filariasis.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-4691 |
| Date | 31 May 2011 |
| Creators | Enright, Tracy |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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