A Ruiz1, M H Kuehn, J L Andorf, E Stone, G S Hageman, D Bok. 1. Department of Neurobiology, Brain Research Institute and. Jules Stein Eye Institute, School of Medicine, University of California, Los Angeles, CA 90095, USA.
Abstract
PURPOSE: To determine the structure of the human lecithin retinol acyltransferase (LRAT) gene, map its chromosomal localization, and screen for mutations in humans with various hereditary retinal degenerations. METHODS: Using DNA probes specific for LRAT, a bacterial artificial chromosome (BAC) clone containing the LRAT gene was isolated, subcloned into DNA fragments and relevant subclones characterized by sequencing. Exon-intron junctions were determined by comparison with the cDNA sequence previously published. Southern blot analysis was performed on human genomic DNA samples digested with several restriction enzymes. Fluorescence in situ hybridization (FISH) analysis of normal metaphase chromosomes derived from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes and radiation hybrid mapping were used for localization of the LRAT gene. Single-strand conformation polymorphism analysis (SSCP) was used to screen for potential mutations in patients with age-related macular degeneration, Leber congenital amaurosis, retinitis pigmentosa, and cone-rod dystrophy. RESULTS: The human LRAT gene is organized into three exons of 219, 541, and 2058 bp and two introns of 103 and 4117 bp. Southern blot analysis of digested genomic DNA revealed a single band, suggesting a single copy of the LRAT gene. The human LRAT gene was localized to chromosome 4q31.2, a locus having no previous association with human eye disease. Additionally, the bovine LRAT homologue sequence was deduced and a general LRAT protein topology is suggested. No polymorphisms that segregated with retinal disease phenotypes were identified in 374 unrelated probands. CONCLUSIONS: The organization of the LRAT gene, based on cDNA clones derived from the retinal pigment epithelium (RPE) has been determined. Its structure is less complex than other acyltransferases such as lecithin cholesterol acyltransferase (LCAT) and acyl CoA acyltransferase (ACAT). The absence of polymorphisms in the probands examined suggests a very low mutation level in the LRAT gene from the diseases analyzed.
PURPOSE: To determine the structure of the humanlecithin retinol acyltransferase (LRAT) gene, map its chromosomal localization, and screen for mutations in humans with various hereditary retinal degenerations. METHODS: Using DNA probes specific for LRAT, a bacterial artificial chromosome (BAC) clone containing the LRAT gene was isolated, subcloned into DNA fragments and relevant subclones characterized by sequencing. Exon-intron junctions were determined by comparison with the cDNA sequence previously published. Southern blot analysis was performed on human genomic DNA samples digested with several restriction enzymes. Fluorescence in situ hybridization (FISH) analysis of normal metaphase chromosomes derived from phytohemagglutinin (PHA) stimulated peripheral blood lymphocytes and radiation hybrid mapping were used for localization of the LRAT gene. Single-strand conformation polymorphism analysis (SSCP) was used to screen for potential mutations in patients with age-related macular degeneration, Leber congenital amaurosis, retinitis pigmentosa, and cone-rod dystrophy. RESULTS: The humanLRAT gene is organized into three exons of 219, 541, and 2058 bp and two introns of 103 and 4117 bp. Southern blot analysis of digested genomic DNA revealed a single band, suggesting a single copy of the LRAT gene. The humanLRAT gene was localized to chromosome 4q31.2, a locus having no previous association with humaneye disease. Additionally, the bovineLRAT homologue sequence was deduced and a general LRAT protein topology is suggested. No polymorphisms that segregated with retinal disease phenotypes were identified in 374 unrelated probands. CONCLUSIONS: The organization of the LRAT gene, based on cDNA clones derived from the retinal pigment epithelium (RPE) has been determined. Its structure is less complex than other acyltransferases such as lecithin cholesterol acyltransferase (LCAT) and acyl CoA acyltransferase (ACAT). The absence of polymorphisms in the probands examined suggests a very low mutation level in the LRAT gene from the diseases analyzed.
Authors: Matthew L Batten; Yoshikazu Imanishi; Tadao Maeda; Daniel C Tu; Alexander R Moise; Darin Bronson; Daniel Possin; Russell N Van Gelder; Wolfgang Baehr; Krzysztof Palczewski Journal: J Biol Chem Date: 2003-12-18 Impact factor: 5.157
Authors: Dean Bok; Alberto Ruiz; Orna Yaron; Wan Jin Jahng; Arghya Ray; Linlong Xue; Robert R Rando Journal: Biochemistry Date: 2003-05-27 Impact factor: 3.162