Cloning and characterization of the mammalian homolog of the Drosophila melanogaster retinal degeneration B gene

Purpose. To clone and characterize the mammalian homolog of the Drosophila melanogaster retinal degeneration B (rdgB) gene. RdgB mutations cause a light-enhanced retinal degeneration in Drosophila. The rdgB gene product is a membrane-associated phosphatidylinositol transferase. Methods. A single-strand DNA-based subtractive hybridization approach was used to generate a subtracted bovine retina/RPE library. RT-PCR was used to determine tissue specificity. In situ hybridization is being used to determine the spatial and temporal expression pattern of the mammalian rdgB homolog (m-rdgB). Results. The subtracted library was tested and found to be highly enriched for retina and/or RPE specific genes. Of the first 400 clones that were sequenced, approximately 25% represent genes that are known to be differentially expressed in the RPE and/or retina. Among the novel genes, a 1.3 kb cDNA clone showed 56% identity and 75% similarity to the Drosophila rdgB gene. A pair of primers was generated based on the sequence from the bovine clone. These primers amplify a 950 bp RT-PCR product from bovine retina and RPE, but not from heart or liver. In addition, these primers amplify a band of about 550 bp from mouse retina and brain, but not from other tissues tested. Cloning of full length bovine and mouse m-rdgBs is underway. The longest bovine clone obtained so far is approximately 3 kb. To identify the cell type specificity and subcelluar localization of m-rdgB, in situ hybridization and immunohistochemistry with mouse tissues are in progress. Conclusions. A mammalian homolog of rdgB exists and is highly expressed in the retina/RPE, and to a lesser extent in brain. It is not detectable in other tissues tested. Further studies will hopefully elucidate the functions of m-rdgB and its possible role in human retinal degeneration.