Abstract
Abstract: :
Purpose: The myopia 2 (MYP2) locus is associated with autosomal dominant 1 high–grade myopia, and has been mapped to chromosome 18p11.31 by genetic linkage analysis. Further identification of the gene(s) has been hindered, as the region is poorly characterized. To facilitate characterization of the MYP2 gene, a detailed transcript map was constructed of the refined mapped interval using bioinformatic tools and exon–trapping strategies for isolation of transcribed sequences from cloned human genomic DNA. Methods: A physical map was compiled of the 4 Mb region on chromosome 18p11.31 using public databases. Known and hypothetical genes were identified. Human genomic BAC clones covering the region were identified and aligned, and 8 overlapping clones were chosen. The BAC clones were subcloned into pSPL3 expression vector and transfected into COS7 cells. Expressed genes were isolated from the cells and cloned into pBluescripts KS(+). Clones were sequenced and aligned against human genomic sequences. Results: The 4 Mb region was covered by 30 BAC clones, and 8 spanning 1.5 Mb were chosen in our initial study. Within the region, there are 6 known and 5 hypothetical genes based on public databases. Various gene prediction programs identify 66 – 83 predicted genes. Among 1000 clones analyzed, many were false positive clones from expression vectors. Remaining clones with unique sequences were analyzed and BLASTED against known sequence databases to reveal putative exons. Expressed putative exons are confirmed by Northern blotting and reverse transcription– polymerase chain reaction techniques. Conclusions: Using a combination of bioinformatics and genetic analysis of isolated exon sequences from human genomic DNA, we constructed a detailed transcript map of the chromosome 18p11.31 MYP2 interval. These results will facilitate the identification of the MYP2 gene(s).
Keywords: gene mapping • gene/expression • myopia