Abstract
Abstract: :
Purpose:The Rd4 model mouse inherits an autosomal dominant retinal degeneration that cosegregates with a large inversion encompassing nearly all of chromosome 4. It was reported that the proximal breakpoint of this inversion is in the centromere itself and that the distal breakpoint maps to the subtelomeric region. We analyzed the distal breakpoint to isolate a candidate gene responsible for the retinal degeneration in the Rd4 mouse. Methods:To define the distal breakpoint region, we employed fluorescence in situ hybridization (FISH) using bacterial artificial chromosome (BAC) DNAs and genomic DNA fragments prepared by PCR. We compared mRNA levels and the developmental expression patterns of a candidate gene in Rd4/+ and C57BL/6J normal retinas by Northern blot analysis and real–time quantitative PCR. Also the level of the gene product protein was examined by Western blot. Results:We previously reported that the distal breakpoint localizes to a 50 kb–interval on the BAC clone, RP23–183d11, which is disrupted by the breakpoint. According to the NCBI database, the gene Gnb1, coding transducin ß–subunit (Gß1), is mapped to this interval. Further FISH analysis using small genomic DNA fragments showed that the breakpoint lies in intron 2 of this gene. The gene consists of 12 exons and 11 introns. The in–frame ATG is located at exon 3, indicating that the breakpoint interrupts the 5’–UTR of the gene. mRNA expression analysis revealed that the mRNA level of Gnb1 in Rd4/+ retina drops to 50 % of wild type retina prior to the beginning of retinal degeneration. The Gß1 (protein) level in Rd4/+ retina also decreases to half of that in wild type retina. Conclusions:These results suggest that Gnb1 is a candidate gene for Rd4 and provide a chance to screen mutations of human Gnb1, which is mapped on human 1p36, using the DNA of patients affected with different retinal degenerations.
Keywords: candidate gene analysis • positional cloning • retinal degenerations: hereditary