11-cis-retinal and Chromosome-Deletion

Topics: Base Sequence; Chromosome Deletion; Codon; Family; Genes, Dominant; Germany; Humans; Molecular Sequence Data; Recurrence; Retinitis Pigmentosa; Rhodopsin

Detection of small alterations or abnormalities in genomic DNA (eg, point mutations or small deletions) has become increasingly important in the diagnosis of genetic disease and polymorphism. When a mutation or polymorphism creates a new restriction endonuclease site, it can easily be identified by polymerase chain reaction (PCR) amplification of the DNA region of interest, followed by digestion with the restriction endonuclease. However, useful restriction sites are the exception, and a variety of specialised techniques have been developed to identify subtle DNA abnormalities. We have shown that where a DNA mutation does not create a useful novel restriction site, such a site can be introduced by PCR and specially chosen primers. The approach is simple and inexpensive and should be broadly applicable in the diagnosis of genetic polymorphism and mutation. The technique is illustrated here by the three base-pair deletion responsible for most cases of cystic fibrosis and by detection of the point mutation in the rhodopsin gene that has been associated with some cases of autosomal dominant retinitis pigmentosa.

Topics: Alleles; Base Sequence; Blood Proteins; Calgranulin A; Chromosome Deletion; Cystic Fibrosis; DNA, Single-Stranded; Evaluation Studies as Topic; Humans; Molecular Sequence Data; Mutation; Oligonucleotide Probes; Polymerase Chain Reaction; Polymorphism, Restriction Fragment Length; Restriction Mapping; Retinitis Pigmentosa; Rhodopsin

To define the cis-acting DNA elements required for rhodopsin expression, we generated lines of transgenic mice carrying sequences upstream of the bovine rhodopsin gene fused to the E. coli beta-galactosidase gene (lacZ). Upstream sequences extending from -2174 to +70 bp, from -734 to +70 bp, and from -222 to +70 bp direct photoreceptor-specific expression. All three -2174 lines demonstrate a superior-temporal to inferior-nasal gradient of expression across the retina, whereas lines carrying the shorter constructs demonstrate either spatially continuous expression across the retina, discrete clusters of expression, or both. As a complementary approach to defining regulatory elements, we compared DNA sequences 5' of the murine, bovine, and human rhodopsin genes. Significant homology between all three species was found just upstream of the transcription start site and at approximately 1.5 kb upstream.

Topics: Aging; Amino Acid Sequence; Animals; beta-Galactosidase; Cattle; Chromosome Deletion; Cloning, Molecular; Escherichia coli; Gene Expression Regulation; Humans; Mice; Mice, Transgenic; Molecular Sequence Data; Photoreceptor Cells; Promoter Regions, Genetic; Retina; Rhodopsin; Sequence Homology, Nucleic Acid

Autosomal dominant retinitis pigmentosa (ADRP) has recently been linked to locus D3S47 (probe C17), with no recombination, in a single large Irish family. Other ADRP pedigrees have shown linkage at zero recombination, linkage with recombination, and no linkage, demonstrating genetic heterogeneity. The gene encoding rhodopsin, the rod photoreceptor pigment, is closely linked to locus D3S47 on chromosome 3q. A point mutation changing a conserved proline to histidine in the 23d codon of the gene has been demonstrated in affected members of one ADRP family and in 17 of 148 unrelated ADRP patients. We have sequenced the rhodopsin gene in a C17-linked ADRP family and have identified in the 4th exon and in-frame 3-bp deletion which deletes one of the two isoleucine monomers at codons 255 and 256. This mutation was not found in 30 other unrelated ADRP families. The deletion has arisen in the sequence TCATCATCAT, deleting one of a run of three x 3-bp repeats. The mechanism by which this occurred may be similar to that which creates length variation in so-called mini- and microsatellites. Thus ADRP is an extremely heterogeneous disorder which can result from a range of defects in rhodopsin and which can have a locus or loci elsewhere in the genome.

Topics: Amino Acid Sequence; Base Sequence; Chromosome Deletion; Chromosomes, Human, Pair 3; DNA Probes; Electrophoresis, Polyacrylamide Gel; Female; Genes, Dominant; Genetic Linkage; Humans; Male; Molecular Sequence Data; Nucleic Acid Hybridization; Pedigree; Recombination, Genetic; Retinitis Pigmentosa; Rhodopsin

Rhodopsin is a member of a family of receptors that contain seven transmembrane helices and are coupled to G proteins. The nature of the interactions between rhodopsin mutants and the G protein, transduction (Gt), was investigated by flash photolysis in order to monitor directly Gt binding and dissociation. Three mutant opsins with alterations in their cytoplasmic loops bound 11-cis-retinal to yield pigments with native rhodopsin absorption spectra, but they failed to stimulate the guanosine triphosphatase activity of Gt. The opsin mutations included reversal of a charged pair conserved in all G protein-coupled receptors at the cytoplasmic border of the third transmembrane helix (mutant CD1), replacement of 13 amino acids in the second cytoplasmic loop (mutant CD2), and deletion of 13 amino acids from the third cytoplasmic loop (mutant EF1). Whereas mutant CD1 failed to bind Gt, mutants CD2 and EF1 showed normal Gt binding but failed to release Gt in the presence of guanosine triphosphate. Therefore, it appears that at least the second and third cytoplasmic loops of rhodopsin are required for activation of bound Gt.

Topics: Amino Acid Sequence; Animals; Cell Line; Cell Membrane; Chromosome Deletion; Micelles; Models, Molecular; Molecular Sequence Data; Mutation; Photolysis; Protein Binding; Protein Conformation; Rhodopsin; Transducin; Transfection

Topics: Chromosome Deletion; Color Vision Defects; Humans; Male; Rhodopsin