Abstract
The fish retinal ganglion cells can successfully recover their axons after optic nerve transection. We searched molecules to trigger the initiation of optic nerve regeneration during the early stage of this process with molecular cloning technique. By using differential hybridization between normal and axotomized retinas, we cloned a full-length cDNA for goldfish purpruin, a secretory 22 kDa retinol-binding protein from the retinal cDNA library. The levels of purpruin mRNA was upregulated in the photoreceptor layers 2-5 days after optic nerve injury. The retinol-binding protein with retinol showed a priming action on neurite outgrowth in the goldfish retina (Matsukawa, et al., 2004). In the present study, in order to test transcriptional regulation of prupurin gene, we do cloning of cDNA and genomic DNA for zebrafish purpurin using the goldfish cDNA probe. The amino acid sequence of zebrafish purpurin cDNA was 94% homology to that of goldfish. We further investigated purpurin mRNA levels in the zebrafish retina by Northern blot hybridization following optic nerve injury. The cellular localization of this mRNA was observed by in situ hybridization. After screening of zebrafish genomic DNA library using goldfish purpurin cDNA, we clone a 10 kbp zebrafish genomic DNA. From the data of sequence analysis, the genomic DNA had six exons and exon-intron boundaries. We are now in progress to do luciferase reporter assay for analysis of promoter regions in the 5’upstream of coding regions of prupurin gene. [Jpn J Physiol 55 Suppl:S163 (2005)]
