In vivo promoter analysis in zebrafish of the Fugu rubripes NMDA receptor subunit 1 gene.

摘要

A 5kb fragment of the pufferfish Fugu rubripes NR1 promoter spanning nucleotides -2729 to +2343 was shown to be sufficient to drive expression of the NR1 gene in zebrafish. I performed a cross-species sequence comparison of the 5kb Fugu NR1 promoter with homologous pufferfish (Tetraodon nigroviridis), zebrafish (Danio rerio ) and stickleback (Gasterosteus aculeatus) genomes and identified five non-coding evolutionary conserved regions (ECRs) containing cis-elements that serve as putative binding sites for eleven different trans-acting factors. I performed 5' serial deletions of the 5kb NR1 fragment based on the location of the ECRs and subsequently injected the truncated promoter constructs into fertilized zebrafish embryos to assess the activity of the deletion constructs.;The results suggest the presence of a putative fish NR1 gene control region that spans nucleotides -1360 to -194 and contains ECRs 1--3. The results also demonstrate that the minimal cis promoter spans nucleotides -149 to +131 and contains ECR4 and ECR5.;Analysis of the NR1 promoter requires an investigation of development in an intact organism. Thus, the main aim of my thesis was to establish a transgenic line of zebrafish expressing the 5kb Fugu NR1 promoter. The transgenic data validated results obtained by in vitro methods regarding the activation of the NR1, which occurs via promoter de-repression, and the regulation of the NR1 gene, whereby tissue-specific trans-acting factors act on its cis-elements and determine its expression.