Cloning of the cDNA for a Novel Photoreceptor Protein

Abstract

A subtractive cDNA cloning strategy was used to isolate a 1381-base pair human retina-specific cDNA, human retinal gene 4 (HRG4), which hybridized to a 1.4-kilobase message in the retina and encoded a 240-amino acid acidic protein with a calculated molecular mass of 26,964 Da. The proximal 1 ⁄4 of the conceptual protein sequence was rich in glycine (18%) and proline (20%), had a predicted secondary structure of turns, and showed a loose similarity (19 -24%) to various ␣-collagen sequences, while the distal 3 ⁄4 consisted of a mixture of ␣-helices, ␤-sheets, and turns. Genomic Southern analysis with HRG4 showed cross-hybridizing sequences in six different species, and HRG4 was 92% homologous with a 1264-base pair rat cDNA (rat retinal gene 4; RRG4) at the protein level. The region of 100% identity between the two sequences corresponded to the distal 3 ⁄4 of the protein sequence consisting of mixed secondary structures, suggesting a functionally important domain. Isolation and characterization of mammalian genes specifically or abundantly expressed in the retina have contributed greatly toward the understanding of retinal biology and disease. Such genes include rhodopsin, cGMP-phosphodiesterase, transducin, and arrestin that are involved in phototransduction (1-4); peripherin and rom-1 involved in photoreceptor disc structure and function (5, 6); and interphotoreceptor retinol binding protein involved in transport of retinol between the retinal pigment epithelium (RPE) 1 and photoreceptors (7). Candidate gene analyses of these genes in inherited retinal degenerations have identified rhodopsin, peripherin, cGMPphosphodiesterase ␤, and rom-1 (possibly by itself and in combination with peripherin) to be implicated in retinopathies including retinitis pigmentosa and macular pattern dystrophies (8 -16).In order to expand our knowledge of retinal biology and to identify additional retinal genes that may be involved in retinal diseases, we have been isolating new retinal genes by a subtractive cDNA cloning strategy. This strategy has already resulted in the isolation and characterization of the cDNA and gene for human recoverin, a protein previously thought to be involved in the recovery of the depolarized state in phototransduction (17), and X-arrestin, a new retinal arrestin that appears to play a desensitization role in a yet to be defined retina-specific signal transduction (18). We now report another cDNA obtained by this strategy that represents a new retinaspecific gene that is highly conserved in human and rat and that begins to express an acidic, hydrophilic protein with some similarity to collagen specifically in photoreceptors at the time of outer retinal maturation and continues to express this protein throughout adult life. MATERIALS AND METHODS Subtractive cDNA CloningA retina-enriched cDNA library was prepared as described previously (17). Northern Blot AnalysisHuman Tissue Blot-Total RNA was isolated from human retina, cornea, iris, cultured retinal pigment epithelial cells, and fibroblasts using guanidin...

Molecular approach to annelid regeneration: cDNA subtraction cloning reveals various novel genes that are upregulated during the large‐scale regeneration of the oligochaete, Enchytraeus japonensis

“…An example of this is glutamine synthetase (seq#8), which we isolated in our current screen and has been previously observed to be expressed in Xenopus during neural development (Hatada et al, 1995), regeneration (Tazaki et al, 2005), and following major burn injuries (Abcouwer et al, 1997). Another clone that we identified from our cDNA subtraction analysis, unc-119 (seq#29), which was originally identified as a mutation that affects nematode locomotion (Maduro and Pilgrim, 1995), has been shown to be highly conserved in metazoans (Higashide et al, 1996;Swanson et al, 1998;Maduro et al, 2000), and has also been suggested to play a role in the development of the nervous system of the zebrafish (Manning et al, 2004).…”

Targeted inactivation of synaptic HRG4 (UNC119) causes dysfunction in the distal photoreceptor and slow retinal degeneration, revealing a new function

“…A genomic clone containing the murine orthologue of HRG4 gene was isolated from a 129 SvJ mouse genomic library (Stratagene, La Jolla, CA) by hybridization screening using a rat HRG4 (RRG4) cDNA as probe (Higashide et al, 1996). The 129 SvJ library was used to obtain a mouse gene clone with minimal difference to the target gene of homologous recombination in the embryonic stem (ES) cells of 129 SvJ lineage.…”

“…Two million lambda Fix II clones from the 129 SvJ mouse genomic library (Stratagene, La Jolla, CA) were plated out and screened by hybridization with a rat HRG4 (RRG4) cDNA probe using standard procedures (Higashide et al, 1996;Sambrook and Fritsch, 1989). Positive clones were purified through multiple picking and hybridization screening, and characterized by restriction mapping and DNA sequencing.…”

“…HRG4 (UNC119) is a photoreceptor synaptic protein that was cloned in our laboratory through a subtractive cloning strategy to isolate novel retinal genes that may be candidate retinal degeneration genes (Higashide et al, 1996). HRG4 is homologous to C. elegans neuroprotein UNC119, loss of which causes disorganized neural architecture and paralysis in the worm (Maduro and Pilgrim, 1995).…”

RIBEYE Recruits Munc119, a Mammalian Ortholog of the Caenorhabditis elegans Protein unc119, to Synaptic Ribbons of Photoreceptor Synapses

“…1) is a mammalian ortholog of the Caenorhabditis elegans protein unc119 and essential for normal vision and synaptic transmission at photoreceptor synapses (1)(2)(3). Munc119/RG4 was initially identified by a differential display screen and shown to be expressed at high levels in photoreceptor synapses (1)(2)(3).…”