Gabriele Dekomien1, Jörg Thomas Epplen. 1. Molecular Human Genetics, Ruhr-University, 44780 Bochum, Germany. gabriele.dekomien@ruhr-uni-bochum.de
Abstract
PURPOSE: We describe the cloning, sequence, and mutation analysis of the canine Recoverin (RCV1) gene, a candidate gene for generalized progressive retinal atrophy (PRA). METHODS: The gene was isolated from a genomic lambda Fix II library using an exon 1 probe of the human RCV1 cDNA. Canine RCV1 sequences were identified by subcloning, polymerase chain reaction (PCR), and sequence analysis. Furthermore, selected DNA samples of 22 dog breeds (including all PRA-affected and several representative unaffected dogs from the pedigrees) were screened for mutations and polymorphisms using PCR-SSCP (single strand conformation polymorphism) and sequence analysis. RESULTS: The canine RCV1 gene revealed 3 exons and an open reading frame of 606 bp, potentially coding for a protein of 202 amino acids. The deduced amino acid sequence of the canine RCV1 gene shares 89% identity with the homologous human, 94% with bovine, and 91% identity with the mouse genes. The protein sequence reveals two typical Ca2+-binding EF-hand motifs. In the ORF (open reading frame) of the RCV1 gene a C272A (exon 1) and a C4275A transversion (exon 3) were discovered. These exchanges result in amino acid substitutions (N3K and P202H), but they do not segregate with PRA in the breeds investigated. Additionally, two sequence variations were identified in the 5'-UTR, one in intron 1 and thirteen variations in intron 2 as well as one in the 3'-UTR. CONCLUSIONS: Using intragenic polymorphisms, we excluded the RCV1 gene as a candidate gene for autosomal recessively transmitted (ar) PRA in 16 dog breeds. In addition the RCV1 gene was excluded for presumedly autosomal dominant (ad) PRA in 8 out of the 22 dog breeds investigated.
PURPOSE: We describe the cloning, sequence, and mutation analysis of the canineRecoverin (RCV1) gene, a candidate gene for generalized progressive retinal atrophy (PRA). METHODS: The gene was isolated from a genomic lambda Fix II library using an exon 1 probe of the humanRCV1 cDNA. CanineRCV1 sequences were identified by subcloning, polymerase chain reaction (PCR), and sequence analysis. Furthermore, selected DNA samples of 22 dog breeds (including all PRA-affected and several representative unaffected dogs from the pedigrees) were screened for mutations and polymorphisms using PCR-SSCP (single strand conformation polymorphism) and sequence analysis. RESULTS: The canineRCV1 gene revealed 3 exons and an open reading frame of 606 bp, potentially coding for a protein of 202 amino acids. The deduced amino acid sequence of the canineRCV1 gene shares 89% identity with the homologous human, 94% with bovine, and 91% identity with the mouse genes. The protein sequence reveals two typical Ca2+-binding EF-hand motifs. In the ORF (open reading frame) of the RCV1 gene a C272A (exon 1) and a C4275A transversion (exon 3) were discovered. These exchanges result in amino acid substitutions (N3K and P202H), but they do not segregate with PRA in the breeds investigated. Additionally, two sequence variations were identified in the 5'-UTR, one in intron 1 and thirteen variations in intron 2 as well as one in the 3'-UTR. CONCLUSIONS: Using intragenic polymorphisms, we excluded the RCV1 gene as a candidate gene for autosomal recessively transmitted (ar) PRA in 16 dog breeds. In addition the RCV1 gene was excluded for presumedly autosomal dominant (ad) PRA in 8 out of the 22 dog breeds investigated.
Authors: Tanja Lippmann; Anna Jonkisz; Tadeusz Dobosz; Elisabeth Petrasch-Parwez; Jorg T Epplen; Gabriele Dekomien Journal: Mol Vis Date: 2007-02-07 Impact factor: 2.367